ANGPTL4 oligonucleotides influencing the regulation of the fatty acid metabolism

ABSTRACT

An ANGPTL4 inhibitor consists of an oligonucleotide has 12 to 22 nucleotides. At least one of the nucleotides is modified, and the oligonucleotide hybridizes with a nucleic acid sequence of human and/or muse ANGPTL4 and inhibits the expression of ANGPTL4. A pharmaceutical composition can include the ANGPTL4 inhibitor and a pharmaceutically acceptable carrier, excipient diluent, or a combination of these.

PRIORITY AND CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional Application of U.S. application Ser. No. 17/293,356, filed May 12, 2021, which is a U.S. National Stage Application under 35 U.S.C. § 371 of International Application No. PCT/EP2019/081161, filed Nov. 13, 2019, designating the U.S. and published in the English language as WO 2020/099478 A2 on May 22, 2020, which claims the benefit of European Patent Application No. EP 180246407.1, filed Nov. 13, 2018. Any and all applications for which a foreign or a domestic priority is claimed are identified in the Application Data Sheet filed herewith and are hereby incorporated by reference in their entirety.

SEQUENCE LISTING IN ELECTRONIC FORMAT

The present application is being filed along with an Electronic Sequence Listing as an ASCII text file via EFS-Web. The Electronic Sequence Listing is provided as a file entitled ZACC231001C1SEQLIST.txt, created and last saved on May Feb. 8, 2422, which is 83,772 bytes in size. The information in the Electronic Sequence Listing is incorporated herein by reference in its entirety.

FIELD

The present invention refers to an inhibitor of ANG PTL4 such as an antisense oligonucleotide and a pharmaceutical composition comprising such an inhibitor as well as its use for treating a cardiovascular disease, obesity, diabetes type II, homozygote familial hypercholesterolemia (HoFH), heterozygote familial hypercholesterolemia (HeFH) or dislipidemia.

TECHNICAL BACKGROUND

Disturbed plasma lipids are well-known risk factors in cardiometabolic disease. Successful treatment for elevated LDL-cholesterol has been given since the mid-80's and the following decades focus has broadened towards other lipid classes such as HDL-cholesterol and triglycerides. Epidemiological studies have revealed that increased plasma triacylglycerol (TG) and concomitant remnant cholesterol is an independent risk factor for coronary heart disease (Cullen, 2000). Furthermore, hypertriglyceridemia (HTG) is a hallmark of the metabolic syndrome (MS) and is often accompanied by obesity and insulin resistance (Reaven, 1995). The increased risk of type 2 diabetes and cardiovascular disease (CVD) associated with metabolic syndrome and HTG suggests that maintenance of plasma TG homeostasis is highly desirable.

Patients with severe hypertriglyceridemia can also develop pancreatitis (Athyros, 2002), particularly when TG levels exceed 1000-1500 mg/dl (Tsuang, 2009). As many as 40 different genes are now known to regulate plasma TG (Johansen, 2011), but only a few monogenetic disorders are known to markedly increase TG (Nordestgaard and Varbo, 2014). These comprises FCS which is described in more detail below. Lipolysis is a key step in clearance of TG-rich lipoproteins that takes place on the luminal surface of capillaries of heart, skeletal muscle, and adipose tissues. LPL synthesized in muscle and adipocytes is translocated to capillary endothelial cells. Rare genetic defects in lipoprotein lipase (LPL) (Benlian, 1996), the main enzyme responsible for the hydrolysis of TG an lipoproteins, can lead to familial chylomicronemia syndrome (FCS) characterized by plasma TG levels well over >10 mmol. Homozygous defects in apolipoprotein C-II (apoC-II), a key protein activator of LPL, can also lead to a similar hypertriglyceridemic phenotype (Breckenridge, 1978). More recently, defects in GPIHBP1, a protein that links LPL to the surface of endothelial cells (Beigneux, 2007), and mutations in apolipoprotein A-V (ApoA-V) (Ishihara, 2005) have also been described to cause hypertriglyceridemia in humans. Genetic defects in the lipase specific chaperone LMF1 has also been found to promote FCS. Taken together about 2-3:1 000 000 patients have FCS.

Not only LPL activating factors affect the LPL system; loss of function mutations in LPL negative regulators such as apoC3 and ANGPTL4, ANGPTL3 or ANGPTL8 has been shown to promote a favorable plasma lipid profile and a reduced risk for metabolic diseases. ANGPTL4 is a regulator of different lipases and LPL in particular. The protein is unfolding chaperones that break up the dimeric catalytically active form of LPL into inactive monomers which is an immersible event. The ANGPTLs are the only known factors to regulate LPL in this manner, compared to e.g. apoC3 which displaces LPL from lipid substrates. In addition ANGPTL4 affects hepatic lipase and endothelial lipase thus affecting not only the TG moiety of plasma lipids but also LDL-c and HDL-c.

ANGPTL4 is a fasting induced factor expressed also by the liver but to a relevant extent by adipose tissue and skeletal muscle as well, i.e., ANGPTL4 is expressed ubiquitary. The expression of ANGPTL4 is regulated by different stimuli; it is for example induced in the liver by Peroxisome Proliferator-Activator Receptor (PPAR)α, PPARδ and the glucocorticoid receptor (GR), respectively. Animal models deficient for these ANGPTLs show increased LPL activity and decreased plasma lipids and mice with transgenic overexpression for the human variants show the opposite. The findings from animal studies are supported by human deficiency and loss of function mutations which correlates for ANGPTL4 with plasma TG levels and HDL-c. The ANGPTL4 gene shows a link to cardiometabolic diseases.

Thus, information to date provides new insights into the coordinate activities of LPL, GPIHBP1, ANGPTLs and apoA-V in plasma TG homeostasis. Among these factors, ANGPTL4 also regulates plasma cholesterol levels i.e. LDL-c, HDL-c and remnant-c intriguingly without being all dependent on the LDL-receptor which in most cases is non-functional in homozygote familial hypercholesterolemia (HoFH) and heterozygote familial hypercholesterolemia (HeFH). This provides an opportunity for an “all-purpose” plasma lipid drug while targeting ANGPTL4.

ANGPTL4 regulates the activity of the lipoprotein lipase that plays an important role in the intake of free fatty acids into the liver. Dysregulation of lipoprotein lipase can lead to a lipid excess in the ells, which results for example in obesity, diabetes type II or cardiovascular diseases.

ANGPTL4 knock-out mice show reduced triglyceride (TG) level based on increased degradation of very low-density lipoprotein (VLDL) and reduced VLDL production. The cholesterol level is influenced moderately. Food having high lipid level results in ANGPTL4 knock-out mice which are treated with monoclonal antibodies to reduced viability due to lesions of lipogranuloma of the intestinal tissue, the effluent lymphatic system and/or the mesenteric lymph nodes (Desai et at, 2007 PNAS). Humans being heterozygous for the ANGPTL4 variant E40K show significant lower plasma TG level when fasting. Also the high density lipoprotein (HDL) cholesterol levels were significantly higher in E40K heterozygous humans. As the combination of high TG and low HDL cholesterol level leads to an increased risk to suffer from cardiovascular diseases, the reduction or inhibition of ANGPTL4 may reduce the risk. ANGPTL4 null alleles exist in humans, but a pathology comparable to ANGPTL4 knock-out mice has not been identified so far.

Oligonucleotides of the present invention inhibiting the expression of ANGPTL4 reduce for example the plasma lipid level independent of LDL receptor functionality, which is relevant for example for use of the oligonucleotides in treating homozygote familial hypercholesterolemia (HoFH) or heterozygote familial hypercholesterolemia (HeFH), where the LDL receptor is defect.

ANGPTL4 is not only involved in the regulation of the fatty acid metabolism, but it is also involved in influenza infection. ANGPTL4 is for example upregulated by STAT3-mediated mechanism during influenza pneumonia and is a potential biomarker for respiratory infection and pneumonia (Li et al., Cell Reports 10, February 2015).

So far no antisense oligonucleotide exists which is highly efficient in reduction and inhibition, respectively, of ANGPTL4 expression and hybridizes with ANGPTL4 mRNA and/or pre-mRNA. Studies with siRNA to inhibit ANGPTL4 expression showed that in vivo inhibition is only possible if siRNA is packed in suitable packaging material. Even if siRNA is packed the efficiency on the inhibition of mRNA expression can often not be improved.

An oligonucleotide of the present invention is very successful in the inhibition of the expression of ANGPTL4. The mode of action of an oligonucleotide differs from the mode of action of an antibody or small molecule, and oligonucleotides are highly advantageous regarding for example

(i) the penetration into tissues,

(ii) the blocking of multiple functions and activities, respectively, of a target,

(iii) the combination of oligonucleotides with each other or an antibody or a small molecule, and

(iv) the inhibition of intracellular effects which are not accessible or not specifically accessible for an antibody or inhibitable via a small molecule.

SUMMARY

The present invention refers to an ANGPTL4 inhibitor consisting of an oligonucleotide comprising or consisting of for example 12 to 22 nucleotides, 15 to 20 nucleotides, or 15, 16, 17, 18, 19 or 20 nucleotides, wherein at least one of the nucleotides is modified. The ANGPTL4 oligonucleotide hybridizes for example with a nucleic acid sequence of ANGPTL4 of SEQ ID NO.1 (human; NM_139314), ANGPTL4 of SEQ ID NO.2 (human; GRCh38_19_364151_8374373) ANGPTL4 of SEQ ID NO.58 (mouse; NM_020581.2) and/or ANGPTL4 of SEQ ID NO.59 (mouse; GRCm38:17:33773750:33781575), wherein the oligonucleotide inhibits the expression of ANGPTL4. The modified nucleotide is for example selected from the group consisting of a bridged nucleic acid such as LNA, cET, ENA, 2′Fluoro modified nucleotide, 2′O-Methyl modified nucleotide, 2′ O-Methoxyethyl modified nucleotide and a combination thereof.

The ANGPTL4 oligonucleotide of the present invention hybridizes for example with an active area selected from position 1732-1759 (e.g., A24044He, SEQ ID NO.47; A24076He, SEQ ID NO.47) and/or from position 234-261 (e.g. A24102He, SEQ ID NO. 177; A24103He, SEQ ID NO. 178) and/or from position 1264-1293 (e.g. A24110He, SEQ ID NO. 185; A24111He, SEQ ID NO. 186) of SEQ ID NO. 1 and/or from position 2800-2872 (eg. A24083Hi, SEQ ID NO. 158; A24085Hi. SEQ ID NO. 160; A24086Hi, SEQ ID NO. 161; A24087Hi, SEQ ID NO. 162) and/or from position 3415-3442 (e.g. A24089Hi, SEQ ID NO. 164) and/or from position 4968-4994 (e.g. A24097Hi, SEQ ID NO. 172) of SEQ ID NO.2 or a combination thereof. It inhibits the expression of ANGPTL4 for example at a nanomolar or micromolar concentration.

The present invention is further directed to a pharmaceutical composition comprising an ANGPTL4 inhibitor of the present invention and a pharmaceutically acceptable carrier, excipient, dilutant or a combination thereof. The inhibitor and the pharmaceutical composition, respectively, are for use in a method of preventing and/or treating a disorder, where an ANGPTL4 imbalance is involved. Such disorder is for example a cardiometabolic disease, obesity, diabetes such as type 2 diabetes mellitus, hypercholesterolemia, hypertriglyceridemia (HTG), dyslipidemia, pancreatitis, metabolic syndrome, familial chylomicronemia syndrome (FCS), influenza infection and/or cancer. Hypercholesterolemia is for example homozygote familial hypercholesterolemia (HoFH) and heterozygote familial hypercholesterolemia (HeFH), cancer is for example breast cancer, lung cancer, malignant melanoma, lymphoma, skin cancer, bone cancer, prostate cancer, liver cancer, brain cancer, cancer of the larynx, gall bladder, pancreas, testicular, rectum, parathyroid, thyroid, adrenal, neural tissue, head and neck, colon, stomach, bronchi, kidneys, basal cell carcinoma, squamous cell carcinoma, metastatic skin carcinoma, osteo sarcoma, Ewing's sarcoma, reticulum cell sarcoma, liposarcoma, myeloma, giant cell tumor, small-cell lung tumor, islet cell tumor, primary brain tumor, meningioma, acute and chronic lymphocytic and granulocytic tumors, acute and chronic myeloid leukemia, hairy-cell tumor, adenoma, hyperplasia, medullary carcinoma, intestinal ganglioneuromas, Wilm's tumor, seminoma, ovarian tumor, leiomyomater tumor, cervical dysplasia, retinoblastoma, soft tissue sarcoma, malignant carcinoid, topical skin lesion, rhabdomyosarcoma, Kaposi's sarcoma, osteogenic sarcoma, malignant hypercalcemia, renal cell tumor, polycythermia vera, adenocarcinoma, anaplastic astrocytoma, glioblastoma multiform, leukemia, or epidermoid carcinoma.

The ANGPTL4 inhibitor or the pharmaceutical composition of the present invention comprising the ANGPTL4 inhibitor are administered locally or systemically.

All documents cited or referenced herein (“herein cited documents”), and all documents cited or referenced in herein cited documents, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an initial screen of 42 human ANGPTL4-specific antisense oligonucleotides. A control oligonucleotide that does not have sequence complementarity to any human or mouse mRNA (Neg1; SEQ ID NO.57) was included as a control. Human epithelioid cervix carcinoma cells (HeLa) were treated with the respective oligonucleotides at a single concentration of 10 μM for three days. Three days alter start of treatment, cells were lysed and ANGPTL4 and HPRT1 mRNA levels were determined using the QuantiGene Singleplex RNA assay. HNRT1 was used as a housekeeping gene for normalization of ANGPTL4 expression. Residual ANGPTL4-mRNA expression relative to mock-treated cells (“no oligo” set as 1). Triplicate wells, mean+/−SD (FIG. 1 ).

FIG. 2 depicts another screen of human ANGPTL4-specific antisense oligonucleotides in SK-OV3 cells to investigate the residual ANGPTL4 mRNA expression.

FIG. 3 depicts further human ANGPTL4-specific antisense oligonucleotides tested in HeLa cells at a concentration of 10 μM. Four of the tested antisense oligonucleotides show more than 50% knockdown of ANGPTL4 mRNA (equivalent to residual mRNA level of <0.5),

FIG. 4 shows another screen of further ANGPTL4-specific antisense oligonucleotides in SK-OV3 cells to investigate the residual ANGPTL4 mRNA expression.

FIG. 5 depicts HEK-Blue™ hTLR9 cells used to study the stimulation of human TLR9 by monitoring the activation of nuclear factor ‘kappa-light-chain-enhancer’ of activated B-cells (NF-kB)-dependent secreted embryonic alkaline phosphatase (SEAP)-production in vitro. ANGPTL4 oligonucleotides A24022Hi (SEQ ID NO.25), A24023Hi (SEQ ID NO.26), A24071Hi (SEQ ID NO.64) and A24076He (SEQ ID NO.47) as well as controls Neg1 (SEQ ID NO.67) and ODN2006 (5′-TCGTCGTTTTGTCGTTTTGTCGTT-3′ PTO-modified—Invivogen cat. no. tlrl-2006; SEQ ID NO.153) were analyzed.

FIG. 6 shows the human ANGPTL4-specific antisense oligonucleotides A24022Hi (SEQ ID NO. 25), A24023Hi (SEQ ID NO. 26), A24071Hi (SEQ ID NO.54) and A24076He (SEQ ID NO.47) with most potent knockdown efficacy in HeLa and SK-OV3 cells, which were selected for determination of half maximal inhibitory concentration (IC₅₀) values. Primary hepatocytes were treated with the respective ANGPTL4 antisense oligonucleotides at different concentrations (5000 nM, 1000 nM, 200 nM, 40 nM, 8 nM, 1.6 nM) for three days and simultaneously treated with PPAR_(Y) (1 μM). Three days after start of treatment, mRNA expression was analyzed using the QuantiGene Singleplex RNA assay as depicted in FIG. 6 . For graphic representation mock-treated cells were set as 0.32 nM.

FIG. 7 shows a first single dose efficacy screen of the additional ANGPTL4-specific antisense oligonucleotides (ASOs) in primary hepatocytes. 25,000 cells/well were seeded in 96-well collagen I-coated flat bottom plates and treated with the respective ASOs at a final concentration of 5 μM. To induce ANGPTL4 mRNA expression, cells were simultaneously treated with 1 μM PPAR_(Y). As a vehicle control, cells were treated with equal amount of DMSO (“DMSO”). Every 24 h, 70 μl of supernatant was replaced with fresh medium containing PPAR_(Y) as well as the respective ASOs at 5 μM or DMSO. After three days, cells were lysed and human HPRT1 as well as human ANGPTL4 mRNA expression was measured using the QuantiGene RNA Singleplex assay. ANGPTL4-mRNA expression values were normalized to expression of the housekeeping gene HPRT1. Residual ANGPTL4-mRNA expression relative to mock-treated cells (“no oligo” set as 1 (n=42), SD=0.24) is shown. Solid line and dotted lines indicate 70% and 0% knockdown efficacy (upper graph) or 100% HPRT1 expression of mock-treated cells (“no oligo” set as 100 (n=42), SD=35.8, graph below), respectively. Data are represented as mean of triplicate wells +/−SD. Positive control ASOs are marked by an asterix.

FIG. 8 depicts a second single dose efficacy screen of the additional ANGPTL4-specific ASOs in primary hepatocytes. 25,000 cells/well were seeded in 96-well collagen I-coated flat bottom plates and treated with the respective ASOs at a final concentration of 5 μM. To induce ANGPTL4 mRNA expression, cells were simultaneously treated with 1 μM PPAR_(Y). As a vehicle control, cells were treated with equal amount of DMSO (“DMSO”) or left untreated (“−DMSO”). Every 24 h, 70 μl of supernatant was replaced with fresh medium containing PPAR_(Y) as well as the respective ASOs at 5 μM or DMSO or medium only. After three days, cells were lysed and human HPRT1 as well as human ANGPTL4 mRNA expression were measured using the QuantiGene RNA Singleplex assay. ANGPTL4-mRNA expression values were normalized to expression of the housekeeping gene HPRT1. Residual ANGPTL4-mRNA expression relative to mock-treated cells (“no oligo” set as 1 (n=36), SD=0.24) is shown. Solid line and dotted lines indicate 70% and 0% knockdown efficacy (upper graph) or 100% HPRT1 expression of mock-treated cells (“no oligo” set as 100 (n=36), SD=19.3, graph below), respectively. Data are represented as mean of sextuplicate (A24076He and negative control oligonucleotides), nonuplicate (−DMSO) or triplicate (all other ASOs) wells +/−SD. Positive control ASO is marked by an asterix.

FIG. 9 depicts NF-kB-activation in HER-Blue hTLR9 SEAP reporter cells. HER-Blue-hTLR9 cells were seeded in flat-bottom 96-well plates and treated with the indicated oligonucleotides for 24 h. Cell supernatants were harvested after the incubation and incubated for 4 h (FIGS. 9A, 9C and 9D) or 3.5 h (FIG. 9B) with QUANTI-Blue solution. SEAP activity was determined by measurement of the optical density (OD) at 620 nm. Data for ANGPTL4-specific ASOs and neg1 treated cells are depicted as mean of triplicates +/−SD of OD units relative to OD units from cells stimulated with 5000 nM ODN2006 (set to 100%). Data for ODN2006 treated cells are depicted as mean+/−SD of centuplicates (A), as mean+/−SD of triplicates (D) or as the mean+/−SD of the means of triplicates on each plate (B-C).

FIG. 10 shows IC₅₀ determination of selected ANGPTL4 ASOs. 25,000 primary human hepatocytes/well were seeded in 96-well plates and treated with different concentrations (5000 nM, 1000 nM, 200 nM, 40 nM, 8 nM, 1.6 nM) of the respective ASO. Every 24 h, 70 μl of supernatant was replaced with fresh medium containing the respective ASOs at indicated concentrations. After three days, cells were lysed and HPRT1 and ANGPTL4 mRNA expression was measured using the QuantiGene RNA Singleplex assay. ANGPTL4-mRNA expression values were normalized to expression of the housekeeping gene HPRT1. Residual ANGPTL4-mRNA expression relative to mock-treated cells (set as 1 (n=36), SD=0.26). Data are represented as mean of triplicate wells +/−SD.

FIG. 11 shows the efficacy of selected ANGPTL4 ASOs on target gene expression after transfection of cynomolgus hepatocytes. 25,000 primary cynomolgus hepatocytes/well were seeded in 96-well plates and transfected with different concentrations (2 nM, 0.2 nM) of the respective ASO. After 24 h incubation at 37° C., cells were lysed and HPRT1 and ANGPTL4 mRNA expression were measured using the QuantiGene RNA Singleplex assay. ANGPTL4-mRNA expression values were normalized to expression of the housekeeping gene HPRT1. Residual ANGPTL4-mRNA expression relative to mock-treated cells (set as 1 (n=12), SD=0.25) is depicted. Data are represented as mean of triplicate wells +/−SD.

FIG. 12 depicts mouse embryonic fibroblasts (3T3 cells) separately treated with the mouse ANGPTL4-specific antisense oligonucleotides as shown in FIG. 12 at a single concentration of 10 μM. Three days after start of treatment, the mRNA levels were determined by QuantiGene RNA Singleplex assay. Hprt1 was used as a housekeeping gene for normalization of ANGPTL4 expression. Right ANGPTL4 antisense oligonucleotides, which are A24047M (SEQ ID NO.106), A24020M (SEQ ID NO.79), A24017M (SEQ ID NO.76), A24021M (SEQ ID NO.80), A24049M (SEQ ID NO.108), A24018M (SEQ ID NO.77), A24041M (SEQ ID NO.100) and A24010M (SEQ ID NO.69), reduced the normalized ANGPTL4 expression by more than 50%.

FIG. 13 depicts mouse renal carcinoma Renca cells separately treated with the ANGPTL4-specific antisense oligonucleotides as shown in FIG. 13 at a single concentration of 10 μM. After three days the mRNA levels were determined by QuantiGene RNA Singleplex assay. Hprt1 was used as a housekeeping gene for normalization of ANGPTL4 expression. A24020M (SEQ ID NO.79) and A24019M (SEQ ID NO.78) resulted in ANGPTL4 knockdown of more than 50% (equivalent to residual mRNA level of <0.5).

FIG. 14 depicts 9 already tested as well as 24 further mouse ANGPTL4-specific antisense oligonucleotides tested in mouse breast cancer cells 4T1. Cells were treated with 5 μM of respective ANGPTL4 antisense oligonucleotides as shown in FIG. 14 without using a transfection reagent. After three days, cell supernatant was replaced by fresh medium containing 5 μM of the respective ANGPTL4 antisense oligonucleotides and incubated for further three days. Afterwards, the mRNA levels were determined by QuantiGene RNA Singleplex assay. Gapdh was used as a housekeeping gene for normalization of ANGPTL4 expression. 12 of the tested antisense oligonucleotides, which are A24017M (SEQ ID NO.76), A24070M (SEQ ID NO.127), A24020M (SEQ ID NO.79), A24019M (SEQ ID NO.78), A24069M (SEQ ID NO.126), A24021M (SEQ ID NO.80), A24011M (SEQ ID NO.70), A24073M (SEQ ID NO. 130), A24018M (SEQ ID NO.77), A24055M (SEQ ID NO.114), A24010M (SEQ ID NO.69), and A24065M (SEQ ID NO.79) show more than 80% knockdown of ANGPTL4 mRNA (equivalent to residual mRNA level of <0.2).

FIG. 15 shows 21 mouse ANGPTL4-specific antisense oligonucleotides tested in 4T1 cells by treating them with 5 μM of the respective ANGPTL4 antisense oligonucleotide as shown in FIG. 16 . After three days, cell supernatant was replaced by fresh medium containing 5 μM of the respective ANGPTL4 antisense oligonucleotide and incubated for additional three days. Afterwards, the mRNA levels were determined by QuantiGene RNA Singleplex assay. Hprt1 was used as a housekeeping gene for normalization of ANGPTL4 expression. A24047M (SEQ ID NO.106), A24095Mi (SEQ ID NO. 151), A24093Mi (SEQ ID NO. 149), A24020M (SEQ ID NO.79), A24090Mi (SEQ TD NO. 146), and A24082Mi (SEQ ID NO.139) show more than 50% knockdown of ANGPTL4 mRNA (equivalent to residual mRNA level of <0.5).

FIG. 16 depicts A24018M (SEQ ID NO.77), A24019M (SEQ ID NO.78), A24020M (SEQ ID NO.79), A24021M (SEQ ID NO.80), A24047M (SEQ ID NO.106), A24054M (SEQ ID NO.113), A24065M (SEQ ID NO.79), A24070M (SEQ ID NO.127), A24072M (SEQ ID NO.129), A24082M (SEQ ID NO.139) and A24095Mi (SEQ ID NO.151) with most potent knockdown efficacy in 3T3, Renca and 4T1 cells, which were selected for determination of half maximal inhibitory concentration (IC₅₀) values. 4T1 cells were treated with these ANGPTL4 antisense oligonucleotides at different concentrations (5000 nM, 1000 nM, 200 nM, 40 nM, 8 nM, 1.6 nM) for three days. After three days, cell supernatant was replaced by fresh medium containing 5 μM of the respective ANGPTL4 antisense oligonucleotides and incubated for additional three days. Then, mRNA expression was analyzed using the QuantiGene Singleplex RNA assay.

FIG. 17 shows a 1^(st) single dose efficacy screen of additional mouse Angptl4-specific ASOs in 4T1 cells. 2,500 4T1 cells/well were seeded in 96-well flat bottom plates and treated with the respective ASOs at a final concentration of 5 μM. Three days after treatment, cell supernatant was replaced by fresh medium w/ASO and cells were incubated for further three days at 37° C. Afterwards, cells were lysed and mouse Hprt1 and mouse Angptl4 mRNA expression were measured using the QuantiGene RNA Singleplex assay. Angptl4-mRNA expression values were normalized to expression of the housekeeping gene Hprt1. Residual Angptl4-mRNA expression relative to mock-treated cells (“no oligo” set as 1 (n=37), SD=0.21) is shown. Solid line and dotted line indicate 75% and 0% knockdown efficacy (upper graph) or 100% Hprt1 level (graph below) (“no oligo” set as 1 (n=37), SD=0.17), respectively. Data are represented as mean of triplicate wells +/−SD.

FIG. 18 shows a 2^(nd) single-dose screen of mouse Angptl4-specific ASOs in Renca cells. 2,500 Renca cells/well were seeded in 96-well flat bottom plates and treated with the respective ASOs at a final concentration of 5 μM. Three days after treatment, cell supernatant was replaced by fresh medium w/ASO and cells were incubated for further three days at 37° C. Afterwards, cells were lysed and mouse Hprt1 and mouse Angptl4 mRNA expression were measured using the QuantiGene RNA Singleplex assay. Angptl4-mRNA expression values were normalized to expression of the housekeeping gene Hprt1. Residual Angptl4-mRNA expression relative to mock-treated cells (“no oligo” set as 1 (n=37), SD=0.37) is shown. Solid line and dotted line indicate 75% and 0% knockdown efficacy (upper graph) or 100% Hprt1 level (graph below) (“no oligo” set as 1 (n=37), SD=0.36), respectively. Data are represented as mean of triplicate wells +/−SD.

FIG. 19 shows IC₅₀ determination of selected Angptl4 ASOs. 15,000 primary mouse hepatocytes/well were seeded in 96-well flat bottom plates and treated with different concentrations (5000 nM, 1000 nM, 200 nM, 40 nM, 8 nM, 1.6 nM) of the respective ASO. After three days, cells were lysed and Hprt1 and Angptl4 mRNA expression were measured using the QuantiGene RNA Singleplex assay. Angptl4-mRNA expression values were normalized to expression of the housekeeping gene Hprt1. Residual Angptl4-mRNA expression relative to mock-treated cells (set as 1 (n=36), Neg1 is displayed in black. Graph below shows Hprt1 raw values relative to no oligo (set as 100 (n=36), SD=13.0). Data are represented as mean of triplicate wells +/−SD.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a successful inhibitor of ANGPTL4 expression, which is a human or mouse oligonucleotide hybridizing with mRNA and/or pre-mRNA sequences of ANGPTL4 and inhibits the expression and activity, respectively, of ANGPTL4. mRNA comprises only exons of the ANGPTL4 encoding nucleic acid sequence, whereas pre-mRNA comprises exons and introns of the ANGPTL4 encoding nucleic acid sequence. Thus, the oligonucleotides of the present invention represent an interesting and highly efficient tool for use in a method of preventing and/or treating disorders, where the ANGPTL4 expression and activity, respectively, is increased.

In the following, the elements of the present invention will be described in more detail. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.

Throughout this specification and the claims, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated member, integer or step or group of members, integers or steps but not the exclusion of any other member, integer or step or group of members, integers or steps. The terms “a” and “an” and “the” and similar reference used in the context of describing the invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by the context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”, “for example”), provided herein is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

An inhibitor which is an oligonucleotide of the present invention is for example an antisense oligonucleotide (ASO) consisting of or comprising 10 to 25 nucleotides, 12 to 22 nucleotides, 15 to 20 nucleotides or 16 to 18 nucleotides. The oligonucleotides for example consist of or comprise 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides.

The oligonucleotide of the present invention forms for example a gapmer consisting of or comprising a central block of at least 5 nucleotides, i.e., deoxynucleotides and/or ribonucleotides, which is flanked by for example naturally and/or artificially modified nucleotides such as deoxynucleotides and/or ribonucleotides.

The oligonucleotides of the present invention comprise at least one nucleotide which is modified. The modified nucleotide is for example a bridged nucleotide such as a locked nucleic acid (LNA, e.g., 2′,4′-LNA), cET, ENA, a 2′Fluoro modified nucleotide, a 2′O-Methyl modified nucleotide, 2′ O-Methoxyethyl modified nucleotide or a combination thereof. In some embodiments, the oligonucleotide of the present invention comprises one or more nucleotides having the same or different modifications. In addition, the oligonucleotide of the present invention optionally comprises a modified phosphate backbone, wherein the phosphate is for example a phosphorothioate.

The oligonucleotide of the present invention comprises the one or more modified nucleotide at the 3′- and/or 5′-end of the oligonucleotide and/or at any position within the oligonucleotide, wherein modified nucleotides follow in a row of for example 1, 2, 3, 4, 5, or 6 modified nucleotides, or a modified nucleotide is combined with one or more unmodified nucleotides. The following Tables 1 to 4 present examples of ANGPTL4 oligonucleotides comprising modified nucleotides for example LNA which are indicated by (+) and phosphorothioate (PTO) indicated by (*). The ANGPTL4 oligonucleotides consisting of or comprising the sequences of Table 1 or 2 (human), or Table 3 or 4 (mouse) may comprise any other modified nucleotide and/or any other combination of modified and unmodified nucleotides. ANGPTL4 oligonucleotides of Table 1 hybridize with mRNA and/or pre-m RNA of human ANGPTL4:

Seq ID Name Antisense Sequence 5′-3′ Antisense Sequence 5′-3′ with PTO (*) and LNA (+)  3 A24001Hi AGGCTAAGTTAGTGGCC + A*G*G*C*T*A*A*G*T*T*A*G*T*G*G* + C* + C  4 A24002Hi GCCAGATAGGCTAAGTT + G* + C*C*A*G*A*T*A*G*G*C*T*A*A* + G* + T* T  5 A24003He CTGGAAAGAATCGGATC + C* +T*G* + G*A*A*A*G*A*A*T*C*G*G* + A* + T* + C  6 A24004He CGCTGGAAAGAATCGGA + C*G* + C*T*G*G*A*A*A*G*A*A*T* + C* + G* + G* + A  7 A24005Hi GCTAGTCTCGACAGCAG + G*C* + T*A*G*T*C*T*C*G*A*C*A*G*C* + A* + G  8 A24006Hi GATCCTCATGATAGGCC + G* + A*T*C*C*T*C*A*T*G*A*T*A*G*G* + C* + C  9 A24007He GGCCGTCGGAGCACCGC + G* + G*C*C*G*T*C*G*G*A*G*C*A*C*C*G* + C 10 A24008Hi CATGCTGTGGTTCGAGA + C*A* + T*G*C*T*G*T*G*G*T*T*C*G* + A* + G* + A 11 A24009He CGTGCGCCAGGACATTC + C* + G*T*G*C*G*C*C*A*G*G*A*C*A* + T* + T* + C 12 A24010He CGTGCGCCAGGACATT + C* + G* + T*G*C*G*C*C*A*G*G*A*C* + A* + T* + T 13 A24011He TCCGTGCGCCAGGACAT + T*C*C*G*T*G*C*G*C*C*A*G*G*A* + C*A* + T 14 A24012He GGAGTCCGTGCGCCAGG + G*G*A*G*T*C*C*G*T*G*C*G*C*C* + A*G* + G 15 A24013He TGCGCTCCGCGTGTTCG + T* + G*C*G*C*T*C*C*G*C*G*T*G*T*T* + C* + G 16 A24014He TGCGCTCCGCGTGTTC + T* + G*C*G*C*T*C*C*G8C*G*T*G*T* + T* + C 17 A24015He GTGCGCTCCGCGTGTT + G* + T*G*C*G*C*T*C*C*G*C*G*T*G*T* + T 18 A24016Hi GTCGAAATGAGTCTGCA + G* + T* + C*G*A*A*A*T*G*A*G*T*C*T* + G* + C* + A 19 A24017Hi GCACGTCTGGAGGCTCA + G* + C* + A*C*G*T*C*T*G*G*A*G*G*C* + T* + C* + A 20 A24018Hi TTGAGCACGTCTGGAGG + T* + T* + G*A*G*C*A*C*G*T*C*T*G*G* + A* + G* + G 21 A24019Hi GGATCGCAGTGCCGCAA + G* + G* + A*T*C*G*C*A*G*T*G*C*C*G* + C* + C* + A 23 A24020He GCTGAATTCGCAGGTGC + G*C* + T*G*A*A*T*T*C*G*C*A*G*G* + T*G* + C 24 A24021Hi GATCGCAGTGCCGCAA + G* + A* + T*C*G*C*A*G*T*G*C*C*G* + C* + A* + A 25 A24022Hi GTGTTGTAACCTCTTGT + G* T* + C*T*T*G*T*A*A*C*C*T*C*T* + T* + C* + T 26 A24023Hi CCGTGTTGTAACCTCTT + C* + C* + G*T*G*T*T*G*T*A*A*C*C*T* + C* + T* + T 27 A24024He CCTCATGGTCTAGGTGC + C* + C* + T* + C*A*T*G*G*T*C*T*A*G*G* + T* + G* + C 28 A24025He CACCTCATGGTCTAGGT + C*A* + C*C*T8C*A*T*G*G*T*C*T*A*G* + G* + T 29 A24026He GGCGCCTCTGAATTACT + G*G*C*G*C*C*T*C*T*G*A*A*T*T* + A* + C* + T 30 A24027He CGTGGCGCCTCTGAATT + C* + G* + T*G*G*C*G*C*C*T*C*T*G*A* + A* + T* + T 31 A24028He TCGTGGCGCCTCTGAAT + T*C*G*T*G*G*C*G*C*C*T*C*T*G*A* + A* + A* + T 32 A24029He CCGCCTTGTAGGCTTCC + C* + C* + G*C*C*T*T*G*T*A*G*G*C*T*T*C* + C 33 A24030He ACTCGGCGTTGCCATCC + A* + C* + T*C*G*G*C*G*T*T*G*C*C*A*T*C* + C 34 A24031He GCCGTGTCCTCGCCACC + G* + C*C*G*T*G*T*C*C*T*C*G*C*C*A*C* + C 35 A24032He GAGGTCGTGATCCTGGT + G* + A*G*G*T*C*G*T*G*A*T*C*C*T*G* + G* + T 36 A24033He GCGGAGGTCGTGATCCT + G* + C* + G*G*A*G*G*T*C*G*T*G*A*T*C* + C* + T 37 A24034He CCTGCGGAGGTCGTGAT + C*C*T*G*C*G*G*A*G*G*T*C*G*T* + G* + A* + T 38 A24035He CTCTTGGCGCAGTTCTT + C* + T* + C*T*T*G*G*C*G*C*A*G*T*T* + C* + T* + T  39 A24036He GCTCTTGGCGCAGTTCT + G* + C* + T*C*T*T*G*G*C*G*C*A*G*T* + T* + C* + T 40 A24037He GGCTCTTGGCGCAGTTC + G* + G* + C*T*C*T*T*G*G*C*G*C*A*G*T* + T* + C 41 A24038He GGTGCCAAACCACCAGC + G* + G*T*G*C*C*A*A*A*C*C*A*C*C* + A* + G* + C 42 A24039He GGAGCGGAAGTACTGGC + G* + G* + A* + G*C*G*G*A*A*G*T*A*C*T*G* + G* + C 43 A24040He ATGGAGCGGAAGTACTG + A* + T* + G*G*A*G*C*G*G*A*A*G*T* + A* + C* + T* + C 44 A24041He GGATGGAGCGGAAGTAC + G* + G* + A*T*G*G*A*G*C*G*G*A*A*G* + T* + A* + C 45 A24042He GGCTGGATCAACATGGT + G* + G* +C*T*G*G*A*T*C*A*A*C*A*T* + G* + G* + T 46 A24043He ATGTGACTGAGTCCGCC + A* + T* + G*T*G*A*C*T*G*A*G*T*C*C* + G* + C* + C 47 A24044He CAATGTGACTGAGTCCG + C* + A* + A* + T*G*T*G*A*C*T*G*A*G*T* + C* + C* + C 48 A24046He GAACTCTGTGAGCTCCG + G* + A* + A*C*T*C*T*G*T*G*A*G*C*T*C*C* + G 49 A24063Hi CTGGTGGACTAACACAC + C* + T* + G*G*T*G*G*A*C*T*A*A*C*A* + C* + A* + C 50 A24064Hi TGGACCTGACAAGGAGA + T* + G* + G*A*C*C*T*G*A*C*A*A*G*G* + A* + G* + A 51 A24065Hi CACACGGCATGTAAGG + C* + A* + C*A*C*G*G*C*A*T*G*T*A* + A* + G* + G  4 A24066Hi GCCAGATAGGCTAAGTT + G* + C* + C*A*G*A*T*A*G*G*C*T*A*A* + G* + T* + T 52 A24067Hi AGGCTAGTCTCGACAGC + A* + G* + G*C*T*A*G*T*C*T*C*G*A*C* + A* + G* + C  8 A24068Hi GATCCTCATGATAGGCC + G* + A* + T*C*C*T*C*A*T*G*A*T*A*G* + G* + C* + C 53 A24070Hi GATCGCAGTGCCGCAAT + G* + A* + T*C*G*C*A*G*T*G*C*C*G*C* + A* + A* + T 53 A24070Hi GATCGCAGTGCCGCAAT + G*A*T*C*G*C*A*G*T*G*C*C*G*C* + A* + A* + T 54 A24071Hi CGTGTTGTAACCTCTTG + C* + G* + T*G*T*T*G*T*A*A*C*C*T*C* + T* + T* + G 55 A24082Hi GATCGCAGTGCCGCA + G* + A* + T*C*G*C*A*G*T*G*C*C* + G* + C* + A 55 A24073Hi GGATCGCAGTGCCGC + G* + A* + T*C*G*C*A*G*T*G*C*C*G* + C* + A 56 A24074Hi CTGGAAAGAATCGGATC + G* + G* + A*T*C*G*C*A*G*T*G*C* + C* + G* + C  5 A24075He CAATGTGACTGAGTCCG + C* + T* + G*G*A*A*A*G*A*A*T*C*G*G* + A* + T* + C 47 A24076He CAATGTGACTGAGTCCG + C* + A* + A*T*G*T*G*A*C*T*G*A*G*T + C* + C* + G 47 A24077He + C* + A* + A*T*G*T*G*A*C*T*G*A*G* + T* + C* + C* + C 57 Neg1 + C* + G* + T*T*T*A*G*G*C*T*A*T*G*T*A* + C* + T* + T Table 1: List of antisense oligonucleotides hybdridizing with human ANGPLT4 mRNA and/or pre-RNA for example of SEQ ID NO. 1 and/or SEQ ID NO. 2; Neg1 is an oligonucleotide representing a negative control which is not hybridizing with ANGPTL4 of SEQ ID NO. 1 or SEQ ID NO. 2. “He” means “human exonic region”  and is an oligonucleotide primarily hybridizing with mRNA of human ANGPTL4, and “Hi” is an oligonucleotide hybridizing with introns of ANGPTL4 pre-mRNA.

ANGPTL4 oligonucleotides of Table 2 also hybridize with mRNA and/or pre-m RNA of human ANGPTL4:

Seq ID Name Antisense Sequence 5′-3′ Antisense Sequence 5′-3′ with PTO (*) and LNA (+) 154 A24078Hi CACCGGAGAGCATCGA + C* + A* + C*C*G*G*A*G*A*G*C*A*T* + C* + G* + A 155 A24079Hi CACCGCATGTAAGGAAG + C* + A* + C*G*G*C*A*T*G*T*A*A*G*G* + A* + A* + G 156 A24080Hi ACACACGGCATGTAAGG + A* + C* + A*C*A*C*G*G*C*A*T*G*T*A* + A* + G* + G 157 A24081Hi TAGTCTCGACAGCAGGT + T* + A* + G*T*C*T*C*G*A*C*A*G*C*A* + G* + G* + T 158 A24082Hi GGTTCGAGATGAACGGA + G* + G* + T*T*C*G*A*G*A*T*G*A*A*C* + G* + G* + A 159 A24083Hi TCGAGATGAACGGAGA + T* + C* + G*A*G*A*T*G*A*A*C*G*G* + A* + G* + A 160 A24084Hi TCTGCATCGGACACACG + T* + C* + T*G*C*A*T*C*G*G*A*C*A* + A* + C* + G 161 A24085Hi AACTTAGAGAACCGCGA + A* + A* + C*T*T*A*G*A*G*A*A*C*C*G* + C* + G* + A 162 A24086Hi GCCGTGAACTTAGAGAA + G* + C* + C*G*T*G*A*A*C*T*T*A*G*A* + G* + A* + A 163 A24087Hi TTAGAGAACCGCGAGT + T* + T* + A*G*A*G*A*A*C*C*G*C*G* + A* + G* + T 164 A24088Hi TGACCAGGAAGACGCTT + T* + G* + A*C*C*A*G*G*A*A*G*A*C*G* + C* + T* + T 165 A24089Hi TCGAAATGAGTCTGCAC + T* + C* + G*A*A*A*T*G*A*G*T*C*T*G* + C* + A* + C 166 A24090Hi CGTTGAGCACGTCTGGA + C* + G* + T*T*G*A*G*C*A*C*G*T*C*T* + G* + G* + A 167 A24091Hi CGTTGAGCACGTCTGG + C* + G* + T*T*G*A*G*C*A*C*G*T*C* + T* + G* +G 168 A24092Hi CACACGTCCAGTTCTCA + C* + A* + C*A*C*G*T*C*C*A*G*T*T*C* + T* + C* + A 169 A24093Hi GCGGATCGGTCAGGAGT + G* + C* + G*G*A*T*C*G*G*T*C*A*G*G* + A* + G* + T 170 A24094Hi TACCACACACCGTCCTG + T* + A* + C*C*A*C*A*C*A*C*C*G*T*C* + C* + T* + G 171 A24095Hi CCTTGTACCACACACCG + C* + C* + T*T*G*T*A*C*C*A*C*A*C*A* + C* + C* + G 172 A24095Hi TTGTACCACACACCGT + T* + T* + G*T*A*C*C*A*C*A*C*A*C + C* + G* T 173 A24097Hi CTTAACAGTGGATGACC + C* + T* + T*A*A*C*A*G*T*G*G*A*T*G* + A* + C* + C 174 A24098He CGAGGACGGTTTTTAT + C* + G* +A* + G*G8A*C*G*G*T*T*T*T* + T* + A* + T 175 A24099He AGGATCCGCTCAGCTCG + A* + G*G*A*T*C*C*G*C*T*C*A*G*C* + T* + C* + G 176 A24100He TCGTGTGAGGATCCGCT + T* + C* + G*T8G*T*G*A*G*G*A*T*C*C* + G* + C* + T 177 A24101He AATCGGATCACAGTCGT + A* + A* + T*C*G*G*A*T*C*A*C*A*G*T* + C* + G* + T 173 A24102He CTTAACAGTGGATGACC + T* + A* + G*C*A*C*G*G*C*G*G*T*G*G* + C* + G* + G 179 A24103He TAGCACGGCGGTGGCG + T* + A* + G*C*A*C*G*G*C*G*G*T*G* + G* + C* + G 180 A24104He GCGGCGACTTGGACTG + G* + C* + G*G*C*G*A*C*T*T*G*G*A* + C* + T* + G 181 A24105He GGACCCAAAGCGCGGC + G* + G* + A*C*G*C*A*A*A*G*C*G*C* + G* + G* + C 182 A24106He AGGACGCAAAGCGCGG + A* + G* + G*A*C*G*C*A*A*A*G*C*G* + C* + G* + G 183 A24107He GCGGCTGACATTGTGAG + G* + C* + G*G*C*T*G*A*C*A*T*T*G*T* + G* + A* + G 184 A24108He ATAGGCCGTGTCCTCGC + A* + T* + A*G*G*C*C*G*T*G*T*C*C*T* + C* + G* + C 185 A24109He TATAGGCCGTGTCCTCG + T* + A* + T*A*G*G*C*C*G*T*G*T*C*C* + T* + C* + G 186 A24110He AGTACTGGCCGTTGAGG + A* + G* + T*A*C*T*G*G*C*C*G*T*T*G* + A* + G* + G 187 A24111He GAAGTACTGGCCGTTGA + G* + A* + A*G*T*A*C*T*G*G*C*C*G*T* + T* + G* + A 188 A24112He TOTTAAGCTTCTGCCGC + T* + C* +T*T*A*A*G*C*T*T*C*T*G*C* + C* + G* + C 189 A24113He AGTCACCGTCTTTCGTG + A* + G* + G*C*A*C*C*G*T*C*T*T*T*C* + G* + T* + G 190 A24114He CGCCATTGAGGCCAGTC + C* + G* + C*C*A*T*T*G*A*G*G*C*C*A* + G* + T* + C 191 A24115He CTGAGTCCGCCATTGAG + C* + T* + G*A*G*T*C*C*G*C*C*A*T*T* + G* + A* + G 192 A24116He ATTGGCGCCTGCTTGTG + A* + T* + T*G*G*C*G*C*C*T*G*C*T*T* + G* + T* + T 193 A24117He ATACCATTGGCGCCTGC + A* + T* + A*C*C8A*T*T*G*G*C*G*C*C* + T* + G* + C 190 A24118He CGCCATTGAGGCCAGTC + C* + G* + C*C*A*T*T*G*A*G*G*C*C*A* + G* + T* + C 194 A24119He TGAGTCCGCCATTGAGG + T* + G* + A*G*T*C*C*G*C*C*A*T*T*G* + A* + G* + G 191 A24120He CTGAGTCCGCCATTGAG + C* + T* + G*A*G*T*C*C*G*C*C*A*T*T* + G* + A* + G 195 A24121He ACTGAGTCCGCCATTGA + A* + C* + T*G*A*G*T*C*C*G*C*C*A*T* + T* + G* + A 196 A24122HMe TGACTGAGTCCGCCATT + T* + G* + A*C*T*G*A*G*T*C*C*G*C*C* + A* + A* + T 197 A24123He TGTGACTGAGTCCGCCA + T* + G* + T*G*A*C*T*G*A*G*T*C*C*G* + C* + C* + A  46 A24124He ATGTGACTGAGTCCGCC + A* + T* + G*T*G*A*C*T*G*A*G*T*C*C* + G* + C* + C 198 A24125He CCCCGTCAGTCAATGTG + C* + C* + C*C*G*T*C*A*G*T*C*A*A*T* + G* + T* + G 199 A24126He GGTCCCCGTCAGTCAAT + G* + G* + T*C*C*C*C*G*T*C*A*G*T*C* + A* + A* + T  57 Neg1 + C* + G* + T*T*T*A*G*G*C*T*A*T*G*T*A* C* + T* + T 249 R01002 + T* + A* + C*G*C*G*C*G*G*T*T*G*T* + T* + T* + A 250 R01009 + T* + T* + A*G*C*G*C*G*C*G*A*A*T* + A* + T* + G 251 R01014 + C* + G* + A*A*T*A*A*C*C*G*T*C*G*T* + G* + T* + T 252 R01019 + G* + A* + C*T*C*G*T*T*A*A*A*C*C*G* + A* + T* + A Table 2: List of antisense oligonucleotides hybridizing with human ANGPLT4 mRNA and/or pre-mRNA for example of SEQ ID NO. 1 and/or SEQ ID NO. 2; Neg1, R01002, R01009, R01014 and R01019 are oligonucleotides representing negative controls which are not hybridizing with ANGPTL4 of SEQ ID NO. 1 and/or SEQ ID NO. 2. “He” means “human exonic region” and is an oligonucleotide primarily hybridizing with mRNA of human ANGPTL4, and “Hi” is an oligonucleotide hybridizing with introns of ANGPTl4 pre-mRNA. “HMe” indicates an oligonucleotide hybridizing with “human and mouse exonic region” of ANGPTL4.

Oligonucleotides of Table 3 hybridize particularly with mRNA and/or pre-m RNA of mouse ANGPTL4:

Seq ID Name Antisense Sequence 5′-3′ Antisense Sequence 5′-3′ with PTO (*) and LNA (+)  60 A24001M GTTGCACCTAAAGCC + G* + T* + T*G*C*A*C*C*T*A*A*A* + G* + C* + C  61 A24002M ACGGTTGCACCTAAAGC + A* + C* + G*G*T*T*G*C*A*C*C*T*A*A* + A* + G* + C  62 A24003M ACGGTTGCACCTAAAG + A* + C* + G*G*T*T*G*C*A*C*C*T*A* + A* + A* + G  63 A24004M TCACGGTTGCACCTAA + T* + C* + A*C*G*G*T*T*G*C*A*C*C* + T* + T* + A  64 A24005M TTCACGGTTGCACCTA + T* + T* + C*A*C*G*G*T*T*G*C*A*C* + C* + T* + A  65 A24006M ATAAGCGTTTCACGGTT + A* + T* + A*A*G*C*G*T*T*T*C*A*C*G* + G* + T* + T  66 A24007M CATAAGCGTTTCACGGT + C* + A* + T*A*A*G*C*G*T*T*T*C*A*C* + G* + G* + T  67 A24008M CGTAGCTCATAAGCGTT + C* + G* + T*A*G*C*T*C*A*T*A*A*G*C* + G* + T* + T  68 A24009M GCTAGGACTCCGGAAC + G* + C* + T*A*G*G*A*C*T*C*C*G*G* + A* + A* + C  69 A24010M ACGCTAGGACTCCGGAA + A* + C* + G*C*T*A*G*G*A*C*T*C*C*G* + G* + A* + A  70 A24011M AACGCTAGGACTCCG + A* + A* + C*G*C*T*A*G*G*A*C*T* + C* + C* + G  71 A24012M AGCGCATGATTCTGG + A* + G* + C*G*C*A*T*G*A*T*T*C* + T* + G* + G  72 A24013M CGGAGCGCAGCGCATGA + C* + G* + G*A*G*C*G*C*A*G*C*G*C*A* + T* + G* + A  73 A24014M AGCCGCGCATAGCACCA + A* + G* + C*C*G*C*G*C*A*T*A*G*C*A* + C* + C* + A  74 A24015M TAGCCGCGCATAGCAC + T* + A* + G*C*C*G*C*G*C*A*T*A*G* + C* + A* + C  75 A24016M AGTAGCCGCGCATAGCA + A* + G* + T*A*G*C*C*G*C*G*C*A*T* + G* + C* + A  76 A24017M CAGTAGCCGCGCATAGC + C* + A* + G*T*A*G*C*C*G*C*G*C*A*T* + A* + G* + C  77 A24018M CAGTAGCCGCGCATAG + C* + A* + G*T*A*G*C*C*G*C*G*C*A* + T* + A* + G  78 A24019M GCAGTAGCCGCGCATA + G* + C* + A*G*T*A*G*C*C*G*C*G*C* + A* + T* + A  79 A24020M CCTTGCGCGCTCAAA + C* + C* + T*T*G*C*G*C*G*C*T*C* + A* + A* + A  80 A24021M GATGCAAAGCGCGGTG + G* + A* + T*G*C*A*A*A*G*C*G*C*G* + G* + T* + G  81 A24022M AGGATGCAAAGCGCGGT + A* + G* + G*A*T*G*C*A*A*A*G*C*G*C* + G* + G* + T  82 A24023M CCACGTGTTCGCGCAG + C* + C* + A*C*G*T*G*T*T*C*G*C*G* + C* + A* + G  83 A24024M CTCCACGTGTTCGCGCA + C* + T* + C*C*A*C*G*T*G*T*T*C*G*C* + G* + C* + A  84 A24025M TGCGCTCCACGTGTTCG + T* + G* + C*G*C*T*C*C*A*C*G*T*G*T* + T* + C* + G  85 A24026M TGCGCTCCACGTGTTC + T* + G* + C*G*C*T*C*C*A*C*G*T*G* + T* + T* + C  86 A24027M TGCGCTCCACGTGTT + T* + G* + C*G*C*T*C*C*A*C*G*T* + G* + T* + T  87 A24028M ATGCGGCGCTCCAGCG + A* + T* + G*C*G*G*C*G*C*T*C*C*A*G*C* + G  88 A24029M CTGACAAGCGTTACCAC + C* + T* + G*A*C*A*A*G*C*G*T*T*A*C* + C* + A* + C  89 A24030M CCTGACAAGCGTTACCA + C* + C* + T*G*A*C*A*A*G*C*G*T*T*A* + C* + C* + A  90 A24031M CCTGACAAGCGTTAC + C* + C* + T*G*A*C*A*A*G*C*G*T* + T* + A* + C  91 A24032M ATTGTCTAGGTGCGTG + A* + T* + T*G*T*C*T*A*G*G*T*G*C* + G* + T* + G  92 A24033M CCATTGTCTAGGTGCGT + C* + C* + A*T*T*G*T*C*T*A*G*G*T*G* + C* + G* + T  93 A24034M CGTTCAGGCGTCTCTGA + C*G* + T*T*C*A*G*G*C*G*T*C*T*C* + T* + G* + A  94 A25035M AGAGCCGTTCAGGCGT + A*G* + A*G*C*C*G*T*T*C*A*G*G* + C*G* + T  95 A24036M GCTCATTGGCCGTGG + G*C* + T*C*A*T*T*G*G*C*C*G* + T*G* + G  96 A24037M AGTGGAAGTATTGTCCA + A* + G*T*G*G*A*A*G*T*A*T*T*G*T* + C*C* + A  97 A24038M TACGCTCCTGCCGTTGC + T* + A* + C*G*C*T*C*C*T*G*C*C*G*T* + T* + G* + C  98 A24039M TTACGCTCCTGCCGTTG + T* + T* + A*C*G*C*T*C*C*T*G*C*C*G* + T* + T* + G  99 A24040M TTTTTACGCTCCTGCCG + T* + T* + T*T*T*A*C*G*C*T*C*C*T*G* + C* + C* + G 100 A24041M TTTTACGCTCCTGCC + T* + T* + T*T*A*C*G*C*T*C*C*T* + G* + C* + C  101 A24042M AGAGGATAGTAGCGGCC + A* + G*A*G*G*A*T*A*G*T*A*G*C*G*G* + C* + C 102 A24043M ACAAGACGCAGATAGCC + A* + C* + A*A*G*A*C*G*C*A*G*A*T*A* + G* + C* + C 103 A24044M GGCGAGAAGTGATATTC + G* + G* + C*G*A*G*A*A*G*T*G*A*T*A* + T* + T* + C 104 A24045M TAGGCGAGAAGTGATAT + T* + A* + G*G*C*G*A*G*A*A*G*T*G*A* + T* + A* + T 105 A24046M GTAGGCGAGAAGTGAT + G* + T* + A*G*G*C*G*A*G*A*A*G*T* + G* + A* + T 106 A24047M GAGTCCGCCATTAAGG + G* + A* + G*T*C*C*G*C*C*A*T*T*A* + A* + G* + G 107 A24048M ACTGAGTCCGCCATTAA + A* + C* + T*G*A*G*T*C*C*G*C*C*A*T* + T* + A* + A 108 A24049M ATATGACTGAGTCCGCC + A* + T* + A*T*G*A*C*T*G*A*G*T*C*C* + G* + C* + C 109 A24050M AATATGACTGAGTCCGC + A* + T* + T*A*T*G*A*C*T*G*A*G*T* + C* + C* + G* + C 110 A24051M CGCTAGGACTCCGGAA + C* + G*C*T*A*G*G*A*C*T*C*C*G* + G* + A* + A 111 A24052M AACGCTAGGACTCCGGA + A* + A*C*G*C*T*A*G*G*A*C*T*C*C*G* + G* + A 112 A24053M AACGCTAGGACTCCGG + A* + A* + C*G*C*T*A*G*G*A*C*T*C*C* + G* + G 113 A24054M CAACGCTAGGACTCCG + C* + A* + A* + C*G*C*T*A*G*G*A*C*T* + C* + C* + G 114 A24055M GCAACGCTAGGACTCCG + G* + C* + A* + A*C*G*C*T*A*G*G*A*C*T* + C* + C* + G 115 A24056M GTAGCCGCGCATAGC + G* + T* + A*G*C*C*G*C*G*C*A*T* + A* + G* + C 116 A24057M AGTAGCCGCGCATAGC + A* + G* + T*A*G*C*C*G*C*G*C*A*T* + A* + G* + C 117 A25058M AGTAGCCGCGCATAG + A* + G* + T*A*G*C*C*G*C*G*C*A* + T* + A* + G 118 A24059M GCAGTAGCCGCGCATAG + G* + C* + A*G*T*A*G*C*C*G*C*G*C*A* + T* + A* + G 118 A24060M GCAGTAGCCGCGCATAG + G* + C* + A* + G*T*A*G*C*C*G*C*G*C*A* + T* + A* + G 119 A24061M CAGTAGCCGCGCATA + C* + A* + G*T*A*G*C*C*G*C*G*C* + A* + T* + A 120 A24062M CCGCAGTAGCCGCGCAT + C*C*G*C*A*G*T*A*G*C*C*G*C*G* + C* + A* + T 121 A24063M CCTTGCGCGCTCAAAA + C*C* + T*T*G*C*G*C*G*C*T*C*A*A* + A* + A 122 A24064M CCTTGCGCGCTCAAAA + C* + C*T*T*G*C*G*C*G*C*T*C*A* + A* + A* + A  79 A24065M CCTTGCGCGCTCAAA + C* + C* + T*T*G*C*G*C*G*C*T*C* + A* + A* + A 123 A24066M ATGCAAAGCGCGGTGG + A* + T* + G*C*A*A*A*G*C*G*C*G* + G* + T* + G* + G 124 A24067M ATGCAAAGCGCGGTG + A* + T* + G*C*A*A*A*G*C*G*C*G* + G* + T* + G 125 A24068M CGCTCCACGTGTTCG + C*G* + C*T*C*C*A*C*G*T*G*T* + T* + C* + G 126 A24069M GCGCTCCACGTGTTCG + G* + C*G*C*T*C*C*A*C*G*T*G*T* + T* + C* + G 127 A24070M TGAGTCCGCCATTAAGG + T* + G* + A* + G*T*C*C*G*C*C*A*T*T*A* + A* + G* + G 128 A24071M ATGACTGAGTCCGCCA + A*T*G*A*C*T*G*A*G*T*C*C*G* + C* + C* + A 129 A24072M TATGACTGAGTCCGCC + T* + A* + T*G*A*C*T*G*A*G*T*C*C* + G* + C* + C 130 A24073M AATATGACTGAGTCCGC + A* + A* + T* + A*T*G*A*C*T*G*A*G*T* + C* + C* + G* + C 131 A24074M AATATGACTGAGTCCG + A* + A* + T* + A*T*G*A*C*T*G*A*G*T* + C*C* + G 132 A24075Mi CCTAGTAGATGCGCCTA + C* + C* + T*A*G*T*A*G*A*T*G*C*G*C*C* + T* + A 133 A24076Mi TCGTTAGTCTAAGTAGA + T* + C* + G*T*T*A*G*T*C*T*A*A*G*T* + A* + G* + A 134 A24077Mi AGTACTAGACTCGTTAG + A* + G* + T*A*C*T*A*G*A*C*T*C*G*T* + T* + A* + G 135 A24078Mi TCGGCAACCTCCTCTTA + T* + C* + G*G*C*A*A*C*C*T*C*C*T*C*T* + T* + A 136 A24079Mi GCTTTATGTCGGCAACC + G* + C* + T*T*T*A*T*G*T*C*G*G*C*A* + A* + A* + C 137 A24080Mi TTGACTCATGCCATAAC + T* + T* + G*A*C*T*C*A*T*G*C*C*A*T* + A* + A* + C 138 A24081Mi GTTAACGGCTAATAAGA + G* + T* + T*A*A*C*G*G*C*T*A*A*T*A* + A* + G* + A 139 A24082Mi TCTGGTTAACGGCTAAT + T* + C* + T*G*G*T*T*A*A*C*G*G*C*T* + A* + A* + T 140 A24083Mi ACTTTAGCTCCTTATGA + A* + C* + T*T*T*A*G*C*T*C*C*T*T*A* + T* + G* + A 141 A24084Mi GATCGAAACTGTTATGT + G* + A* + T*C*G*A*A*A*C*T*G*T*T* + A* + T* + G* + T 141 A24085Mi AGAACCTATGCCTGCCG + A* + G* + A*A*C*C*T*A*T*G*C*C*T*G*C* + C* + G 142 A24086Mi TCCTCTCATCCAATCGG + T* + C* + C*T*C*T*C*A*T*C*C*A*A*T* + C* + G* + G 143 A24087Mi ACGGCTATGTCTGTTAC + A* + C* + G*G*C*T*A*T*G*T*C*T*G*T* + T* + A* + C 144 A24088Mi GGACCTGTAACCACCTA + G* + G* + A*C*C*T*G*T*A*A*C*C*A*C* + C* + T* + A 145 A24089Mi GTATTGTCGCTGATGAA + G* + T* + A*T*T*G*T*C*G*C*T*G*A*T* + G* + A* + A 146 A24090Mi GATTTGCCTAAACTCGT + G* + A* + T*T*T*G*C*C*T*A*A*A*C*T* + C* + G* + T 147 A24091Mi GAGCTTGCGATGCCTGT + G*A* + G*C*T*T*G*C*G*A*T*G*C*C*T* + G* + T 148 A24092Mi GTAGATGACTAGGCCTG + G* + T* + A*G*A*T*G*A*C*T*A*G*G*C*C* + T* + G 149 A24093Mi TCAATGGAAGCGCTTTA + T* + C* + A*A*T*G*G*A*A*G*C*G*C*T* + T* + T* + A 151 A24094Mi GTAGCAGACTTGCACTA + G* + T* + A*G*C*A*G*A*C*T*T*G*C*A* + C* + T* + A 151 A25095Mi CTCATGTTAGGTAGGTT + C* + T* + C*A*T*G*T*T*A*G*G*T*A*G* + G* + T* + T 152 A24096Mi ACGTGGCCAAAGACAAT + A* + C* + G*T*G*G*C*C*A*A*A*G*A*C* + A* + A* + T  57 Neg1 + C* + G* + T*T*T*A*G*G*C*T*A*T*G*T*A* + C* + T* + T Table 3: List of antisense oligonucleotides hybridizing with mouse ANGPLT4 mRNA and/or pre-mRNA for example of SEQ ID NO. 58 and/or SEQ ID NO. 59; Neg1 is an oligonucleotide representing a negative control which is not hybridizing with ANGPTL4 of SEQ ID NO. 58 or SEQ ID NO. 59. Oligonucleotides primarily hybridizing with mouse ANGPLT4 mRNA are indicated by “M”, and oligonucleotides primarily hybridizing with mouse ANGPLT4 pre-mRNA are indicated by “Mi” as the oligonucleotides hybridize with an intron.

Oligonucleotides of Table 4 also hybridize particularly with mRNA and/or pre-m RNA of mouse ANGPTL4:

Seq ID Name Antisense Sequence 5′-3′ Antisense Sequence 5′-3′ with PTO (*) and LNA (+) 200 A24097M TTCACGGTTGCACCTAA + T* + T* + C*A*C*G*G*T*T*G*C*A*C*C* + T* + T* + A 201 A24098M ATAAGCGTTTCACGGT + A* + T* + A*A*G*C*G*T*T*T*C*A*C* + G* + G* + T 202 A24099M GCTAGGACTCCGGAACG + G* + C* + T*A*G*G*A*C*T*C*C*G*G*A* + A* + C* + T 203 A24100M CAGCAACGCTAGGACTC + C* + A* + G*C*A*A*C*G*C*T*A*G*G*A* + C* + T* + C 204 A24101M GTGCAGCAACGCTAGGA + G* + T* + G*C*A*G*C*A*A*C*G*C*T*A* + G* + G* + A 205 A24102M CCGCGCATAGCACCAGG + C* + C* + G*C*G*C*A*T*A*G*C*A*C*C* + A* + G* + G 206 A24103M ATGCAAAGCGCGGTGGC + A* + T* + G*C*A*A*A*G*C*G*C*G*G*T* + G* + G* + C 207 A24104M CCACGTGTTCGCGCAGC + C* + C* + A*C*G*T*G*T*T*C*G*C*G*C* + A* + G* + C 208 A24105M AGCGTTACCACAGGCAG + A* + G* + C*G*T*T*A*C*C*A*C*A*G*G* + C* + A* + G 209 A24106M TCTATCCTCGGAGTCTT + T* + C* + T*A*T*C*C*T*C*G*G*A*G*T* + C* + T* + T 210 A24107M CTCGAAGTCTTGTCTAC + C* + T* + C*G*A*A*G*T*C*T*T*G*T*C* + T* + A* + C 211 A24108M GAAGTATTGTCCATTGA + G* + A* + A*G*T*A*T*T*G*T*C*C*A*T* + T* + G* + A 212 A24109M CAGAGGATAGTAGCGGC + C* + A* + G*A*G*G*A*T*A*G*T*A*G*C* + G* + G* + C 213 A24110M CAGTCTCTCCAGTTACG + C* + A* + G*T*C*T*C*T*C*C*A*G*T*T* + A* + C* + G 214 A24111M GCGAGAAGTGATATTC + G* + C* + G*A*G*A*A*G*T*G*A*T*A*T* + T* + T* + C 107 A24112M ACTGAGTCCGCCATTAA + A* + C* + T*G*A*G*T*C*C*G*C*C*A*T* + T* + A* + A 108 A24113M ATATGACTGAGTCCGCC + A* + T* + A*T*G*A*C*T*G*A*G*T*C*C* + G* + C* + C 215 A24114M TCAATATGACTGAGTCC + T* + C* + A*A*T*A*T*G*A*C*T*G*A*G* + T* + C* + C 216 A24115M TCCAGTCAGTCAATATG + T* + C* + C*A*G*T*C*A*G*T*C*A*A*T* + A* + T* + G 217 A24116M CTCCAGTCAGTCAATAT + C* + T* + C*C*A*G*T*C*A*G*T*C*A*A* + T* + A* + T 218 A24117Mi GAGTCCTCAGTAGATGCG + G* + A* + G*T*C*C*T*A*G*T*A*G*A*T* + G* + C* + G 219 A24118Mi GTTAGTCTAAGTAGAGT + G* + T* + T*A*G*T*C*T*A*A*G*T*A*G* + A* + G* + T 220 A24119Mi AGTACTAGACTCGTTA + A* + G* + T*A*C*T*A*G*A*C*T*C*G*T* + T* + T* + A 221 A24120Mi GCAACCTCCTCTTATTC + G* + C* + A*A*C*C*T*C*C*T*C*T*T*A* + T* + T* + C 222 A24121Mi AAGATATGCAAGGCTAG + A* + A* + G*A*T*A*T*G*C*A*A*G*G*C* + T* + A* + G 223 A24122Mi GACTCATGCCATAACAA + G* + A* + C*T*C*A*T*G*C*C*A*T*A*A* + C* + A* + A 224 A24123Mi GTGGACCTGACAAGAAG + G* + T* + G*G*A*C*C*T*G*A*C*A*A*G* + A* + A* + G 225 A24124Mi AACGGCTAATAAGATTT + A* + A* + C*G*G*C*T*A*A*T*A*A*G*A* + T* + T* + T 226 A24125Mi GTTCTGGTTAACGGCTA + G* + T* + T*C*T*G*G*T*T*A*A*C*G*G* + C* + T* + A 227 A24126Mi GGTGTGCTTACTCTGGT + G* + T* + T*G*T*G*C*T*T*A*C*T*C*T* + G* + G* + T 228 A24127Mi CCTAGAAATTGTGATCG + C* + C* + T*A*G*A*A*A*T*T*G*T*G*A* + T* + C* + C 229 A24128Mi AACGAATAGGCATGAAC + A* + A* + C*G*A*A*T*A*G*G*C*A*T*G* + A* + A* + C 230 A24129Mi ACTTTCACCTAGTTGGC + A* + C* + T*T*T*C*A*C*C*T*A*G*T*T* + G* + G* + C 231 A24130Mi CTCTACTTGGCTAGGCT + C* + T* + C*T*A*C*T*T*G*G*C*T*A*G* + G* + C* + T 232 A24131Mi GGTACTCTGAATTAGTA + G* + G* + T*A*C*T*C*T*G*A*A*T*T*A* + C* + T* + A 233 A24132Mi TGTAACCACCTAAAGCC + T* + G* + T*A*A*C*C*A*C*C*T*A*A*A* + G* + C* + C 234 A24133Mi AGGTATTGTCGCTGATG + A* + G* + G*T*A*T*T*G*T*C*G*C*T*G* + A* + T* + G 235 A24134Mi GAACAGAGGTATTGTCG + G* + A* + A*C*A*G*A*G*G*T*A*T*T*G* + T* + C* + G 236 A24135Mi CATGTATCACACCTTCC + C* + A* + T*G*T*A*T*C*A*C*A*C*C*T* + T* + C* + C 237 A24136Mi TAAACTCGTTCCTGCCT + T* + A* + A*A*C*T*C*G*T*T*C*C*T*G* + C* + C* + T 238 A24137Mi GCCTAAACTCGTTCCTG + G* + C* + C*T*A*A*A*C*T*C*G*T*T*C* + C* + T* + G 239 A24138Mi ATCTCAGGAGCTTATAC + A* + T* + C*T*C*A*G*G*A*G*C*T*T*A* + T* + A* + C 240 A24139Mi ACAAGCTGCATAATAGG + A* + C* + A*A*G*C*T*G*C*A*T*A*A*T* + A* + G* + G 241 A24140Mi TATCACTGAGCTTGCGA + T* + A* + T*C*A*C*T*G*A*G*C*T*T*G* + C* + G* + A 242 A24141Mi AATGGAAGCGCTTTACC + A* + A* + T*G*G*A*A*G*C*G*C*T*T*T* + A* + C* + C 243 A24142Mi TAGCAGACTTGCACTAT + T* + A* + G*C*A*G*A*C*T*T*G*C*A*C* + T* + A* + T 244 A24143Mi AGGCTCAACTCTCGCAC + A* + G* + G*C*T*C*A*A*C*T*C*T*C*G* + C* + A* + C 245 A24144Mi AAGAGCTAGTACTGTAG + A* + A* + G*A*G*C*T*A*G*T*A*C*T*G* + T* + A* + G 246 A24145Mi TATAATTTGATCCTGAC + T* + A* + T*A*A*T*T*T*G*A*T*C*C*T* + G* + A* + C 247 A24146Mi GGTTCTCTGCCAAATGA + G* + G* + T*T*C*T*C*T*G*C*C*A*A*A* + T* + G* + A 248 A24147Mi CGTGGCCAAAGACAATT + C* + G* + T*G*G*C*C*A*A*A*G*A*C*A* + A* + T* + T 151 A24148Mi CTCATGTTAGGTAGGTT + C* + T* + C*A*T*G*T*T*A*G*G*T*A*G* + G* + T* + T  57 Neg1 + C* + G* + T*T*T*A*G*G*C*T*A*T*G*T*A* + C* + T* + T 249 R01002 + T* + A* + C*G*C*G*C*G*G*T*T*G*T* + T* + T* + A 250 R01009 + T* + T* + A*G*C*G*C*G*C*G*A*A*T* + A* + T* + G 251 R01014 + C* + G* + A*A*T*A*A*C*C*G*T*C*G*T* + G* + T* + T 252 R01019 + G* + A* + C*T*C*G*T*T*A*A*A*C*C*G* + A* + T* + A Table 4: List of antisense oligonucleotides hybridizing with mouse ANGPLT4 mRNA and/or pre-mRNA for example of SEQ ID NO. 58 and/or SEQ ID NO. 59; Neg1, R01002, R01009, R01014 and R01019 are oligonucleotides representing negative controls which are not hybridizing with ANGPTL4 of SEQ ID NO. 58 or SEQ ID NO. 59. Oligonucleotides primarily hybridizing with mouse ANGPLT4 mRNA are indicated by “M”, and oligonucleotides primarily hybridizing with mouse ANGPLT4 pre-mRNA are indicated by “Mi” as the oligonucleotides hybridize with an intron.

The oligonucleotides hybridize for example within a hybridizing active area which is an area enriched for ASOs with high activity. The hybridizing active area is for example one or more region(s) on the ANGPTL4 mRNA, e.g., of SEQ ID NO.1 and/or the ANGPTL4 pre-mRNA, e.g., of SEQ ID NO.2, where hybridization with an oligonucleotide highly likely results in a potent knockdown of the ANGPTL4 expression. In the present invention surprisingly several hybridizing active areas were identified for example selected from hybridizing active areas for example selected from position 1732-1759 (e.g., A24044He, SEQ ID NO.47; A24076He, SEQ ID NO.47) of SEQ ID NO.1 and/or from position 6603-6631 (e.g., A24022Hi, SEQ ID NO.25; A24023Hi, SEQ ID NO.26; A24071Hi, SEQ ID NO.54) of SEQ ID NO.2. Further hybridizing active areas are from position 234-261 (e.g. A24102He, SEQ ID NO.178; A24103He, SEQ ID NO.179) and/or from position 1264-1293 (e.g. A24110He, SEQ ID NO.186; A24111He, SEQ ID NO.187) of human SEQ ID NO.1 and/or from position 2800-2872 (e.g. A24083Hi, SEQ ID NO. 159; A24085Hi, SEQ ID NO.161; A24086Hi, SEQ ID NO.162; A24087Hi, SEQ ID NO. 163) and/or from position 3415-3442 (e.g. A24089Hi, SEQ ID NO.165) and/or from position 4968-4994 (e.g. A24097Hi, SEQ ID NO. 173) of human SEQ ID NO.2. Hybridizing active areas on mouse SEQ ID NO.58 or SEQ ID NO.59 are for example from position 137-163 (e.g. A24054M, SEQ ID NO.113) and/or from position 215-299 (e.g. A24018M, SEQ ID NO.77; A24019M, SEQ ID NO.78; A24020M, SEQ ID NO.79; A24021M, SEQ ID NO.80; A24065M, SEQ ID NO.79) and/or from position 1343-1371 (e.g. A24042M, SEQ ID NO.101; A24109M, SEQ ID NO.212) and/or from position 1738-1771 (e.g. A24047M, SEQ ID NO.106; A24070M, SEQ ID NO. 127; A24072M, SEQ ID NO. 129) of SEQ ID NO.58 and/or from position 1286-1314 (e.g. A24082Mi. SEQ ID NO.139; A24125Mi, SEQ ID NO.226) and/or from position 5485-5511 (e.g. A24095Mi, SEQ ID NO.151; A24148Mi, SEQ ID NO.151) of SEQ ID NO.59.

The oligonucleotide of the present invention inhibits for example at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of ANGPTL4 such as the, e.g., human or mouse, ANGPTL4 expression. The oligonucleotide of the present invention inhibits the expression of ANGPTL4 at a nanomolar or micromolar concentration for example in a concentration range of 0.1 nM to 100 μM, 0.5 nM to 15 nM, 0.6 nM to 10 nM, 1 nM to 10 μM, 5 nM to 5 μM, 10 nM to 1 μM, 15 nM to 950 nM, 20 nM to 900 nM, 25 nM to 850 nM, 30 nM to 800 nM, 35 nM to 750 nM, 40 nM to 700 nM, 45 nM to 6501041, 50 nM to 500 nM, or 40 nM to 150 nM, or in a concentration of 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900 or 950 nM, or 1.10 or 100 μM.

The ANGPTL4 oligonucleotide of the present invention is for example used in a concentration range of 1 nM to 10 μM, 5 nM to 6.6 μM, 10 nM to 5 μM, 15 nM to 3 μM, 20 nM to 2.2 μM, 25 nM to 1 μM, 30 nM to 800 nM, 50 nM to 500 nM, 60 nM to 300 nM, 70 nM to 260 nM, 80 nM to 200 nM, 90 nM to 120 nM, or in a concentration of 1, 1.6, 3, 5, 8, 9, 10, 15, 20, 25, 27, 30, 40, 50, 75, 82, 100, 200, 250, 300, 500, or 740 nM, or 1, 2.2, 3, 5, 6.6 or 10 μM.

The ANGPTL4 oligonucleotide of the present invention is for example administered once or repeatedly, e.g., every 12 h, every 24 h, every 48 h for some weeks, months or years, or it is administered every week, every two weeks, every three weeks or every months or every three or six months.

In some embodiments the present invention refers to a pharmaceutical composition comprising an ANGPTL4 oligonucleotide of the present invention and a pharmaceutically acceptable carrier, excipient and/or dilutant. Optionally, the pharmaceutical composition further comprises a chemotherapeutic, another disease specific active agent such as insulin, angiotensin-converting enzyme inhibitor, angiotensin receptor blacker, another oligonucleotide not of the present invention, an antibody, a HERA fusion protein, a ligand trap, a Fab fragment, a nanobody, a BiTe and/or a small molecule which is for example effective in tumor treatment, treatment of diabetes and its side effects, treatment of a cardiovascular disease, obesity, diabetes type II, hypercholesterolemia such as homozygote familial hypercholesterolemia (HoFH), heterozygote familial hypercholesterolemia (HeFH) or dyslipidemia.

The ANGPTL4 oligonucleotide or the pharmaceutical composition of the present invention is for use in a method of preventing and/or treating a disorder for example a disorder where an ANGPTL4 imbalance is involved. Optionally, the use of the oligonucleotide or the pharmaceutical composition of the present invention in a method of preventing and/or treating a disorder is combined with radiotherapy. The radiotherapy may be further combined with a chemotherapy (e.g., platinum, gemcitabine). The disorder is for example characterized by an ANGPTL4 imbalance, i.e., the ANGPTL4 level is increased in comparison to the level in a normal, healthy cell, tissue, organ or subject. The ANGPTL4 level is for example increased by an increased ANGPTL4 expression and activity, respectively. The ANGPTL4 level is measured by any standard method such as immunohistochemistry, western blot, quantitative real time PCR or QuantiGene assay known to a person skilled in the art.

The ANGPTL4 oligonucleotide or a pharmaceutical composition of the present invention is administered locally or systemically for example orally, sublingually, nasally, subcutaneously, intravenously, intraperitoneally, intramuscularly, intratumoral, intrathecal, transdermal, and/or rectal. Alternatively or in combination ex vivo treated immune cells are administered. The ANGPTL4 oligonucleotide is administered alone or in combination with another ANGPTL4 antisense oligonucleotide of the present invention and optionally in combination with another compound such as another oligonucleotide not of the present invention, an antibody, a HERA fusion protein, a ligand trap, a Fab fragment, a nanobody, a BiTe, a small molecule and/or a chemotherapeutic (e.g., platinum, gemcitabine) and/or another disease specific agent such as insulin, angiotensin-converting enzyme inhibitor, and/or angiotensin receptor blocker.

The oligonucleotide not of the present invention, an antibody, a HERA fusion protein, a ligand trap, a Fab fragment, a nanobody, a BiTe, and/or the small molecule are effective in preventing and/or treating a tumor, influenza infection, diabetes such as diabetes type II and its side effects, a cardiovascular disease, obesity, hypercholesterolemia such as homozygote familial hypercholesterolemia (HoFH), heterozygote familial hypercholesterolemia (HeFH) or dyslipidemia. An ANGPTL4 oligonucleotide or a pharmaceutical composition of the present invention is used for example in a method of preventing and/or treating a solid tumor or a hematologic tumor. Examples of cancers preventable and/or treatable by use of the oligonucleotide or pharmaceutical composition of the present invention are breast cancer, lung cancer, malignant melanoma, lymphoma, skin cancer, bone cancer, prostate cancer, liver cancer, brain cancer, cancer of the larynx, gall bladder, pancreas, testicular, rectum, parathyroid, thyroid, adrenal, neural tissue, head and neck, colon, stomach, bronchi, kidneys, basal cell carcinoma, squamous cell carcinoma, metastatic skin carcinoma, osteo sarcoma, Ewing's sarcoma, reticulum cell sarcoma, liposarcoma, myeloma, giant cell tumor, small-cell lung tumor, islet cell tumor, primary brain tumor, meningioma, acute and chronic lymphocytic and granulocytic tumors, acute and chronic myeloid leukemia, hairy-cell tumor, adenoma, hyperplasia, medullary carcinoma, intestinal ganglioneuromas, Wilm's tumor, seminoma, ovarian tumor, leiomyomater tumor, cervical dysplasia, retinoblastoma, soft tissue sarcoma, malignant carcinoid, topical skin lesion, rhabdomyosarcoma, Kaposi's sarcoma, osteogenic sarcoma, malignant hypercalcemia, renal cell tumor, polycythermia vera, adenocarcinoma, anaplastic astrocytoma, glioblastoma multiforma, leukemia, or epidermoid carcinoma.

Further examples of diseases preventable and/or treatable by use of the ANGPTL4 oligonucleotide or the pharmaceutical composition of the present invention other than cancer are for example diabetes such as diabetes type II and its side effects, a cardiovascular disease, obesity, hypercholesterolemia such as homozygote familial hypercholesterolemia (HoFH), heterozygote familial hypercholesterolemia (HeFH) or dyslipidemia.

In some examples two or more ANGPTL4 oligonucleotides of the present invention are administered together, at the same time point for example in a pharmaceutical composition or separately, or on staggered intervals. In some examples two or more ANGPTL4 oligonucleotides of the present invention are administered together, at the same time point for example in a pharmaceutical composition or separately, or on staggered intervals. In other examples, one or more oligonucleotides of the present invention are administered together with another compound such as another oligonucleotide not of the present invention, an antibody, a HERA fusion protein, a ligand trap, a Fab fragment, a nanobody, a BiTe, a small molecule and/or a chemotherapeutic, at the same time point for example in a pharmaceutical composition or separately, or on staggered intervals.

A subject of the present invention is for example a mammalian, a bird or a fish.

EXAMPLES

The following examples illustrate different embodiments of the present invention, but the invention is not limited to these examples. The following experiments are performed on cells endogenously expressing ANGPTL4, i.e., the cells do not represent an artificial system comprising transfected reporter constructs. Such artificial systems generally show a higher degree of inhibition and lower IC₅₀ values than endogenous systems which are closer to therapeutically relevant in vivo systems. Further, no transfecting agent is used in the following experiments, i.e., gymnotic delivery is performed. Transfecting agents are known to increase the activity of an oligonucleotide which influences the IC₅₀ value (see for example Zhang et al., Gene Therapy, 2011, 18, 326-333; Stanton et al., Nucleic Acid Therapeutics, Vol. 22, NO. 5, 2012). As artificial systems using a transfecting agent are hard or impossible to translate into therapeutic approaches and no transfection formulation has been approved so far for oligonucleotides, the following experiments are performed without any transfecting agent, except for the experiments of Example 11, where a transfection reagent was used.

Example 1: 1^(st) Screen of Human ANGPTL4-Specific Antisense Oligonucleotides in HeLa Cells

5,000 HeLa cells/well were seeded in 96-well plates and treated with the respective antisense oligonucleotides as indicated in FIG. 1 at a final concentration of 10 μM. To induce ANGPTL4 mRNA expression, cells were simultaneously treated with 1 μM PPARδ (Sigma Aldrich, Cat. No. SML1491). PPARδ used in the example is for example based on a PPARδ stock solution (10 mM) that was prepared by dissolving 5 mg PPARδ (Molecular weight: 453.50) in 1.1 ml DMSO. For final concentration of 1 μM PPARδ, cells seeded in 96-well plates were incubated with 100 μl medium supplemented with 0.01 μl PPARδ stock solution. As a negative control, cells were treated with equal volume of DMSO. Three days after start of treatment, cells were lysed and human HPRT1 as well as human ANGPTL4 mRNA expression was measured using the QuantiGene RNA Singleplex assay (FIG. 1 ). The QuantiGene Assay used in the examples is for example built upon the branched DNA technology (bDNA), which relies on cooperative hybridization between a target mRNA and a specific probe set (part of QuantGene Reagent System). The assay is performed according to manufacturer's protocol (Thermo Fisher Scientific) and is used for determination of RNA levels. It combines the QuantiGene Sample Processing Kit that is used for cell lysis and the QuantiGene Reagent System that is used for hybridization, amplification and detection of RNA of interest. The QuantiGene Reagent System is based on an RNA-specific probe set, designed to detect a particular RNA of interest.

ANGPTL4-mRNA expression values were normalized to expression of the housekeeping gene HPRT1. Residual ANGPTL4-mRNA expression relative to mock-treated cells (“no oligo” set as 1) is shown. Solid line and dotted lines indicate 70% and 50% or 0% knockdown efficacy, respectively. Data are represented as mean of triplicate wells +/−SD.

As shown in FIG. 1 , two antisense oligonucleotides (A24022Hi (SEQ ID NO.25) and A24023Hi (SEQ ID NO.26)) reduced the normalized ANGPTL4 expression by more than 70% (equivalent to residual mRNA level of <0.3). Further, three antisense oligonucleotides (A24003He (SEQ ID NO.5), A24042He (SEQ ID NO.45), A24005Hi (SEQ ID NO.7)) showed knockdown efficacy between 70% and 50%, while control oligonucleotide (Neg1) did not reduce ANGPTL4 mRNA expression.

Example 2: 2^(nd) Screen of Human ANGPTL4-Specific Antisense Oligonucleotides in SK-OV3 Cells

5,000 SK-OV3 cells/well were seeded in 96-well plates and treated with the respective ANGPTL4 antisense oligonucleotides at a final concentration of 10 μM. To induce ANGPTL4 mRNA expression, cells were simultaneously treated with 1 μM PPARδ (Sigma Aldrich, cat. no. SML1491; for preparation of 10 mM stock solution see example 1). For final concentration of 1 μM PPARδ, cells seeded in 96-well plates were incubated with 100 μl medium supplemented with 0.01 μl PPARδ stock solution. As a negative control, cells were treated with equal volume of DMSO.

Three days after start of treatment, cells were lysed and human HPRT1 as well as human ANGPTL4 mRNA expression was measured using the QuantiGene RNA Singleplex assay. ANGPTL4-mRNA expression values were normalized to expression of the housekeeping gene HPRT1. Residual ANGPTL4-mRNA expression relative to mock-treated cells (“no oligo” set as 1) is shown in FIG. 2 . Solid line and dotted lines indicate 60% and 0% knockdown efficacy, respectively. Data are represented as mean of triplicate wells +/−SD.

Repetition of the ANGPTL4 antisense oligonucleotide screening in SK-OV3 cells resulted in three ANGPTL4 antisense oligonucleotides (A24022Hi (SEQ ID NO.25), A24044He (SEQ ID NO.47), A24023Hi (SEQ ID NO.26)) with more than 60% knockdown efficiency.

Example 3: 1^(st) Single Dose Efficacy Screen of Further Human ANGPTL4-Specific Antisense Oligonucleotides in HeLa Cells

5,000 HeLa cells/well were seeded in 96-well plates and treated with the most efficient ANGPTL4 antisense oligonucleotides (A24022Hi (SEQ ID NO.25), A24023Hi (SEQ ID NO.26), A24044He (SEQ ID NO.47)) from 1^(st) screenings in HeLa (FIG. 1 ) and SK-OV3 (FIG. 2 ) as well as further ANGPTL4-specific antisense oligonucleotides (A24071Hi (SEQ ID NO.54), A24076He (SEQ ID NO.47), A24075He (SEQ ID NO.5), A24073Hi (SEQ ID NO.55), A24065Hi (SEQ ID NO.51), A24067Hi (SEQ ID NO.52), A24077He (SEQ ID NO.47), A24074Hi (SEQ ID NO.56)) at a final concentration of 10 μM. To induce ANGPTL4 mRNA expression, cells were simultaneously treated with 1 μM PPARδ (Sigma Aldrich, cat. no. SML1491; for preparation of 10 mM stock solution see example 1). For final concentration of 1 μM PPARδ, cells seeded in 96-well plates were incubated with 100 μl medium supplemented with 0.01 μl PPARδ stock solution. As a negative control, cells were treated with equal volume of DMSO.

Three days after start of treatment, cells were lysed and human HPRT1 as well as human ANGPTL4 mRNA expression was measured using the QuantiGene RNA Singleplex assay. ANGPTL4-mRNA expression values were normalized to expression of the housekeeping gene HPRT1. Residual ANGPTL4-mRNA expression relative to mock-treated cells (“no oligo” set as 1) is shown in FIG. 3 . Solid line and dotted lines indicate 50% and 0% knockdown efficacy, respectively. Data are represented as mean of triplicate wells +/−SD.

As shown in FIG. 3 , four of the tested ANGPTL4 antisense oligonucleotides (A24022Hi (SEQ ID NO.25), A24023Hi (SEQ ID NO.26), A24044He (SEQ ID NO.47), A24071Hi (SEQ ID NO.54)) show more than 50% knockdown of ANGPTL4 mRNA (equivalent to residual mRNA level of <0.5), while treatment with Neg1 negative control oligonucleotide did not result in decreased ANGPTL4 mRNA levels.

Example 4: 2^(nd) Single Dose Efficacy Screen of Further Human ANGPTL4-Specific Antisense Oligonucleotides in SK-OV3 Cells

5,000 SK-OV3 cells/well were seeded in 96-well plates and treated with the respective ANGPTL4 antisense oligonucleotides (A24071Hi (SEQ ID NO.54), A24022Hi (SEQ ID NO.25), A24076He (SEQ ID NO.47), A24044He (SEQ ID NO.47), A24023Hi (SEQ ID NO.26), A24077He (SEQ ID NO.47), A24075He (SEQ ID NO.5), A24073Hi (SEQ ID NO.55)) at a final concentration of 10 μM. To induce ANGPTL4 mRNA expression, cells were simultaneously treated with 1 μM PPARδ. (Sigma Aldrich, cat. no. SML1491; for preparation of 10 mM stock solution see example 1). For final concentration of 1 μM PPARδ, cells seeded in 96-well plates were incubated with 100 μl medium supplemented with 0.01 μl PPARδ stock solution. As a negative control, cells were treated with equal volume of DMSO.

Three days after start of treatment, cells were lysed and human HPRT1 as well as human ANGPTL4 mRNA expression was measured using the QuantiGene RNA Singleplex assay. ANGPTL4-mRNA expression values were normalized to expression of the housekeeping gene HPRT1. Residual ANGPTL4-mRNA expression relative to mock-treated cells (“no oligo” set as 1) is shown in FIG. 4 . Solid line and dotted lines indicate 50% and 0% knockdown efficacy, respectively. Data are represented as mean of triplicate wells +/−SD.

Repetition of the optimized ANGPTL4-specific antisense oligonucleotide screening in SK-OV3 cells resulted in five ANGPTL4 antisense oligonucleotides (A24071Hi (SEQ ID NO.54), A24022Hi (SEQ ID NO.25), A24076He (SEQ ID NO.47), A24044He (SEQ ID NO.47), A24023Hi (SEQ ID NO.26)) with more than 50% knockdown efficiency (FIG. 4 ).

Example 5: In Vitro TLR9 Assay of Selected Human ANGPTL4-Specific Antisense Oligonucleotides

Binding of immune stimulatory ligands, e.g. bacterial DNA or immune stimulatory oligonucleotides with or without unmethylated CpG dinucleotides results in TLR activation. As immune activation can lead to severe, possibly life threatening condition of excessive cytokine release, there is an urgent need for a preclinical test system that predicts cytokine release in humans.

HEK-Blue-hTLR9 (Invivogen cat. no. hkb-htlr9) cells were seeded in flat-bottom 96-well plates and treated with ANGPTL4 oligonucleotides A24022Hi (SEQ ID NO. 25), A24023Hi (SEQ ID NO. 26), A24071Hi (SEQ ID NO.54) and A24076He (SEQ ID NO.47) for 24 h. Then, cell supernatants were harvested and incubated for 4 h with QUANTI-Blue solution (Invivogen cat. no. rep-qbs). SEAP activity was determined by measurement of the optical density. Means and standard deviations of OD units relative to OD units from cells stimulated with 5000 nM ODN2006 (set as 100) are depicted in FIG. 5 . Data are represented as mean of triplicate wells +/−SD.

As shown in FIG. 5 , none of the tested ANGPTL4-specific antisense oligonucleotides induced TLR9 activation. In contrast, positive control CpG oligonucleotide ODN2006 (5′-TCGTCGTTTTGTCGTTTTGTCGTT-3′ PTO-modified—Invivogen cat. no. tlrl-2006; SEQ ID NO.153) clearly stimulated NFκB activation (FIG. 5 ).

Example 6: IC₅₀ Determination of Selected Human ANGPTL4-Specific Antisense Oligonucleotides

30,000 primary human hepatocytes/well were seeded in 96-well plates and treated with different ANGPTL4 antisense oligonucleotides in different concentrations of 5000 nM, 1000 nM, 200 nM, 40 nM, 8 nM and 1.6 nM: A24022Hi (SEQ ID NO.25), A24023Hi (SEQ ID NO.26), A24071Hi (SEQ ID NO.54) and A24076He (SEQ ID NO.47). To induce ANGPTL4 mRNA expression, cells were simultaneously treated with 1 μM PPAR_(Y). (Sigma Aldrich, cat. no. R2408). PPAR_(Y) stock solution (10 mM) was prepared by dissolving 10 mg PPAR_(Y) (Molecular weight 357.43) in 2.8 ml DMSO. For final concentration of 1 μM PPAR_(Y), cells seeded in 96-well plates were incubated with 100 μl medium supplemented with 0.01 μl PPAR_(Y) stock solution. As a negative control, cells were treated with equal volume of DMSO. Every 24 h, 70 μl of supernatant was replaced with fresh medium containing 1 μM PPAR_(Y) as well as the respective ANGPTL4 antisense oligonucleotides at indicated concentrations. Three days after start of treatment, cells were lysed and HPRT1 and ANGPTL4 mRNA expression was measured using the QuantiGene RNA Singleplex assay. ANGPTL4-mRNA expression values were normalized to expression of the housekeeping gene HPRT1. Residual ANGPTL4-mRNA expression relative to mock-treated cells (set as 1). For graphic representation mock-treated cells were set as 0.32 nM. Data are represented as mean of triplicate wells +/−SD.

FIG. 6 and Table 5 demonstrate that the selected ANGPTL4-specific antisense oligonucleotides inhibit ANGPTL4 mRNA expression dose-dependently with IC₅₀ values in the nanomolar range.

TABLE 5 IC₅₀ values of selected human ANGPTL4-specific antisense oligonucleotides determined in primary human hepatocytes. ASO IC50 [nM] R square A24022Hi 20 0.82 A24023Hi 25 0.83 A24071Hi 16 0.83 A24076He 66 0.76

Example 7: 1^(st) Single-Dose Screens of Additional Human ANGPTL4-Specific Antisense Oligonucleotides (ASOs) in Primary Hepatocytes

In total further 49 ANGPTL4-specific antisense-oligonucleotides were designed. Based on two initial screens in human cell lines (data not shown), 17 promising ASOs were selected for first screen in primary human hepatocytes (FIG. 7 ). Three control oligonucleotide (R01002, R01014, Neg1) with different lengths (16, 17 and 18 nucleotides, respectively) that do not have sequence complementarity to any human or mouse mRNA were included as negative controls, while three ANGPTL4-specific oligonucleotides with verified knockdown efficiency were used as positive controls (A24022Hi (SEQ ID NO.25), A24071Hi (SEQ ID NO.54), A24076He (SEQ ID NO.47)). Human primary hepatocytes (Lonza) were treated with the respective oligonucleotides at a single concentration of 5 μM for three days without the use of a transfection reagent. To induce ANGPTL4 mRNA expression, cells were simultaneously treated with 1 μM PPAR_(Y) (Sigma Aldrich, cat. no. R2408; for preparation of 10 mM stock solution see example 6). For final concentration of 1 μM PPAR_(Y), cells seeded in 96-well plates were incubated with 100 μl medium supplemented with 0.01 μl PPAR_(Y) stock solution. As a negative control, cells were treated with equal volume of DMSO.

Three days after start of treatment, cells were lysed and mRNA levels were determined by QuantiGene RNA Singleplex assay. Hypoxanthine phosphoribosyltransferase 1 (HPRT1) was used as a housekeeping gene for normalization of ANGPTL4 expression. As shown in FIG. 7 , PPAR_(Y) stimulation resulted in ˜50% increased ANGPTL4 levels (no oligo) compared to DMSO-treated cells.

Treatment with eight ANGPTL4-specific ASOs (A24096Hi (SEQ ID NO.172), A24103He (SEQ ID NO.179), A24091Hi (SEQ ID NO.167), A24123He (SEQ ID NO.197), A24083Hi (SEQ ID NO.159), A24087Hi (SEQ ID NO.163), A24102He (SEQ ID NO.178), A24116He (SEQ ID NO.192)) as well as with the positive control ASOs (A24022Hi (SEQ ID NO.25), A24071 Hi (SEQ ID NO.54), A24076He (SEQ ID NO.47)) reduced ANGPTL4 expression by more than 70% (equivalent to residual ANGPTL4-mRNA expression of <0.3). Control oligonucleotides (Neg1, R01002, R01014) did not reduce ANGPTL4 mRNA expression.

Example 8: 2^(nd) Single-Dose Screens of Additional Human ANGPTL4-Specify Antisense Oligonucleotides (ASOs) in Primary Hepatocytes

Remaining newly designed ANGPTL4-specific ASOs were tested in primary hepatocytes under the experimental conditions of Example 7 (FIG. 8 ). These ASOs were well tolerated in a subsequent in vitro test (data not shown). One ANGPTL4-specific oligonucleotide with verified knockdown efficiency was used as positive control (A24076He (SEQ ID NO.47)), while three control oligonucleotides (R01009, R01019, Neg1) with different lengths (16, 17 and 18 nucleotides, respectively), that do not have sequence complementarity to any human or mouse mRNA were included as negative controls.

Treatment with ten ANGPTL4-specific ASOs (A24083Hi (SEQ ID NO.159), A24089Hi (SEQ ID NO.165), A24117He (SEQ ID NO.193), A24124He (SEQ ID NO.46), A24103He (SEQ ID NO.179), A24097Hi (SEQ ID NO.173), A24110He (SEQ ID NO.186), A24121He (SEQ ID NO.195), A24086Hi (SEQ ID NO.162), A24085Hi (SEQ ID NO.161)) reduced ANGPTL4 expression by more than 70% (equivalent to residual ANGPTL4-mRNA expression of <0.3), while incubation with positive control ASO A24076H decreased ANGPTL4 mRNA expression by about 63% (FIG. 8 ). Control oligonucleotides (Neg1, R01009, R01019) only slightly reduced ANGPTL4 mRNA expression (by 6-20%).

Example 9: Activation of Human Toll-Like Receptor 9 (hTLR9) in Response to Human Angiopoietin-Like Protein 4 (ANGPTL4)-Specify LNA-Modified Antisense Oligonucleotides

A potential of human ANGPTL4-specify LNA-modified antisense oligonucleotides A24076H (SEQ ID NO.47), A24083Hi (SEQ ID NO.159), A24085Hi (SEQ ID NO.161), A24086Hi (SEQ ID NO.162), A24087Hi (SEQ ID NO.163), A24089Hi (SEQ ID NO.165), A24096Hi (SEQ ID NO.172), A24102He (SEQ ID NO.178), A24103He (SEQ ID NO.179), A24110He (SEQ ID NO.186), A24111He (SEQ ID NO.187), A24113He (SEQ ID NO.189), A24116He (SEQ ID NO.192) and A24123He (SEQ ID NO.197) were tested to activate TLR9. The experiment was conducted once (A24076H, A24096H, A24102He, A24113He, A24116He and A24123He) or twice (all other ASOs) in a TLR9 reporter cell line HEK-Blue-hTLR9 cells (Invivogen cat. no. hkb-htlr9) under the experimental conditions of Example 5. As shown in FIGS. 9A, 9B, 9C and 9D, no dose-dependent activation of NF-κB upon treatment of the cells with different concentrations of the respective human ANGPTL4 ASO was observed. In contrast, NF-κB was activated in a dose-dependent manner upon treatment with the positive control ODN2006 (5′-TCGTCGTTTTGTCGTTTTGTCGTT-3′ PTO-modified—Invivogen cat. no. tlrl-2006; SEQ ID NO. 153).

Example 10: IC₅₀ Determination of Selected Human ANGPTL4-Specific Antisense Oligonucleotides (ASOs)

Based on knockdown efficiency in primary hepatocytes (FIGS. 7 and 8 ), ASOs with most potent knockdown efficacy and no induction of caspase3/7 upon transfection (data not shown) were selected for determination of IC₅₀ values. As a positive control ANGPTL4-specific ASO A24076He (SEQ ID NO.47) with verified knockdown efficiency was used.

Primary human hepatocytes (Primacyt) were treated with ANGPTL4-specific ASOs or negative control oligonucleotides Neg1, R01009 and R01019 at different concentrations for three days. Simultaneously, cells were treated with PPAR_(Y) (1 μM) (Sigma Aldrich, cat. no. R2408; for preparation of 10 mM stock solution see example 6) to induce ANGPTL4 expression. For final concentration of 1 μM PPAR_(Y), cells seeded in 96-well plates were incubated with 100 μl medium supplemented with 0.01 μl PPAR_(Y) stock solution. As a negative control, cells were treated with equal volume of DMSO. After three days, mRNA expression was analyzed using the QuantiGene Singleplex RNA assay.

Table 6 and FIG. 10 demonstrate that the eight ANGPTL4-specific ASOs (A24083Hi (SEQ ID NO.159), A24085Hi (SEQ ID NO.161), A24087Hi (SEQ ID NO.163), A24089Hi (SEQ ID NO.165), A24097Hi (SEQ ID NO.173), A24103He (SEQ ID NO.179), A24110He (SEQ ID NO.186) and A24111He (SEQ ID NO.187)) and the positive control A24076He (SEQ ID NO.47) inhibit ANGPTL4 mRNA expression dose-dependently with IC₅₀ values in the nanomolar range. Treatment with ASO A24086Hi (SEQ ID NO.162) did not result in dose-dependent reduction of ANGPTL4 mRNA expression (R squared=0.5). Therefore, data were not included.

Following Table 6 shows IC₅₀ values and R squares of selected human ANGPTL4-specific antisense oligonucleotides determined in primacy human hepatocytes. *, R square below 0.85.

ASO IC50 [nM] R square A24083Hi   28 0.95 A24085Hi.   62 0.87 A24087Hi   45 0.92 A24089Hi ~34 0.84* A24097Hi   90 0.65* A24102He   36 0.71* A24110He   12 0.93 A24111He   58 0.73* A24076He   99 0.87

Example 11: In Vitro Efficacy of Human ANGPTL4-Specific Oligonucleotides in Cynomolgus Hepatocytes

In vivo tolerated human ANGPTL4-specific antisense oligonucleotides A24076H (SEQ ID NO.47) as well as three further human ANGPTL4-specific ASOs having only 1 mismatch to cynomolgus ANGPTL4 sequence (Table 7) were tested in primary cynomolgus hepatocytes (FIG. 11 ). All ASOs tested were shown to have verified knockdown efficiency in human cell lines and primary hepatocytes. Two control oligonucleotides (R01009, R01019) with different lengths (16 and 17 nucleotides, respectively), that do not have sequence complementarity to any human or mouse RNA were included as negative controls.

Table 7 shows human ANGPTL4-specific ASOs with proven knock-down efficiency in human cells that do not lead to caspase 3/7 induction in vitro. Depicted are cross-reactivity (CroseReact) as well as number of mismatches to cynomolgus (Mfa, Macaca fascicularis) ANGPTL4 sequence, as well as activity in primary cynomolgus hepatocytes in vitro (FIG. 11 ):

Mis- Activity SEQ matches in Mfa ID to Mfa hepato- Target ASO ID NO. CrossReact_MFA sequence cytes ANGPTL4 A24076He  47 yes 0 + A24083Hi 159 no 4 NA A24085Hi 161 no 4 NA A24086Hi 162 no 6 NA A24087Hi. 163 no 3 NA A24089Hi 165 no 1 + A24097Hi 173 no 3 NA A24103He 179 no 5 NA A24110He 186 no 1 + A24111He 187 no 1 +

Primary cynomolgus hepatocytes (Primacyt) were transfected with ANGPTL4-specific ASOs or negative control oligonucleotides R01009 and R01019 at different concentrations for three days. Simultaneously, cells were treated with PPARδ (1 μM) (Sigma Aldrich, cat. no. R2408; for preparation of 10 mM stock solution see example 6) to induce ANGPTL4 expression. Far final concentration of 1 μM PPAR_(Y), cells seeded in 96 well plates were incubated with 100 μl medium supplemented with 0.01 μl PPAR_(Y) stock solution. As a negative control, cells were treated with equal volume of DMSO. After three days, mRNA expression was analyzed using the QuantiGene Singleplex RNA assay.

As shown in FIG. 11 , treatment with PPARδ induced ANGPTL4 expression about 8-fold (FIG. 11 , DMSO control). Treatment with the ANGPTL4-specific ASO A24076He (SEQ ID NO.47), which is completely cross-reactive to cynomolgus ANGPTL4 sequence led to mare than 70% knock-down of ANGPTL4 (equivalent to residual mRNA expression of 0.3). Human ANGPTL4-specific oligonucleotides having one mismatch to cynomolgus sequence (A24089Hi (SEQ ID NO.165), A24110He (SEQ ID NO.186), A24111He (SEQ ID NO.187)) also reduced cynomolgus ANGPTL4 mRNA up to 51% (equivalent to residual ANGPTL4-mRNA expression of 0.49). Negative control oligonucleotide R01009 did not reduce Angptl4 expression in primary cynomolgus hepatocytes, while control oligonucleotide R01019 only slightly decreased ANGPTL4 mRNA expression by about 25% (equivalent to residual ANGPTL4-mRNA expression of 0.75) using a concentration of 2 nM.

Conclusion Based on Example 11:

Taken together, the in vitro experiments led to the identification of highly potent human ANGPTL4 specific ASOs that are eligible for testing of an ASO-based ANGPTL4-targeting therapeutic in cynomolgus monkeys. Such a drug is used for example for the systemic treatment of dyslipidemia patients in order to reduce the ANGPTL4-mediated inhibition of lipoprotein lipase L preventing cellular lipid overload, obesity, diabetes type II and cardiovascular disease.

Example 12: 1^(st) Single-Dose Screen of Mouse ANGPTL4-Specific Antisense Oligonucleotides in 3T3 Cells

4,500 3T3 cells/well were seeded in 96-well plates and treated with the ANGPTL4 antisense oligonucleotides as shown in FIG. 12 at a final concentration of 10 μM. Cells were lysed and mouse Hprt1 and mouse ANGPTL4 mRNA expression was measured using the QuantiGene RNA Singleplex assay. ANGPTL4-mRNA expression values were normalized to expression of the housekeeping gene Hprt1. Residual ANGPLT4-mRNA expression relative to mock-treated cells (“no oligo” set as 100) is shown in FIG. 12 . Solid line and dotted line indicate 50% and 0% knockdown efficacy, respectively. Data are represented as mean of triplicate wells +/−SD.

As shown in FIG. 12 , eight ANGPTL4 antisense oligonucleotides, i.e., A24047M (SEQ ID NO.106), A24020M (SEQ ID NO.79), A24017M (SEQ ID NO.76), A24021M (SEQ ID NO.80), A24049M (SEQ ID NO.108), A24018M (SEQ ID NO.77), A24041M (SEQ ID NO. 100) and A24010M (SEQ ID NO.69), reduced the normalized ANGPTL4 expression by more than 50%, while control oligonucleotide (Neg1) did not reduce ANGPTL4 mRNA expression (FIG. 12 ).

Example 13: 2^(nd) Single-Dose Efficacy Screen of Mouse ANGPTL4-Specific Antisense Oligonucleotides in Renca Cells

5,000 Renca cells/well were seeded in 96-well plates and treated with the ANGPTL4 antisense oligonucleotides as shown in FIG. 13 at a final concentration of 10 μM. Three days after start of treatment, cells were lysed and mouse Hprt1 and mouse ANGPTL4 mRNA expression was measured using the QuantiGene RNA Singleplex assay. ANGPTL4-mRNA expression values were normalized to expression of the housekeeping gene Hprt1. Residual ANGPTL4-mRNA expression relative to mock-treated cells (“no oligo” set as 1) is shown. Solid line and dotted line indicate 50% and 0% knockdown efficacy, respectively. Data are represented as mean of triplicate wells +/−SD. As shown in FIG. 13 , treatment with two ANGPTL4-specific antisense oligonucleotides, which are A24020M (SEQ ID NO.79) and A24019M (SEQ ID NO.78) resulted in ANGPTL4 knockdown of more than 50% (equivalent to residual mRNA level of <0.5) (FIG. 13 ).

Example 14: Test of Further Mouse ANGPTL4-Specific Antisense Oligonucleotides in 4T1 Cells

2,500 4T1 cells/well were seeded in 96-well plates and treated with the ANGPTL4 antisense oligonucleotides (ASO) as shown in FIG. 14 at a final concentration of 5 μM. After 3 d, cell supernatant was replaced by fresh medium w/ASO. Six days after start of treatment, cells were lysed and mouse Gapdh and mouse ANGPTL4 mRNA expression were measured using the QuantiGene RNA Singleplex assay. ANGPTL4-mRNA expression values were normalized to expression of the housekeeping gene Gapdh. Residual ANGPTL4-mRNA expression relative to Neg1-treated cells (set as 1) is shown. Solid line and dotted lines indicate 80% and 0% knockdown efficacy, respectively. Data are represented as mean of triplicate wells +/−SD.

As shown in FIG. 14 , 12 of the tested antisense oligonucleotides, which are A24017M (SEQ ID NO.76), A24070M (SEQ ID NO.127), A24020M (SEQ ID NO.79), A24019M (SEQ ID NO.78), A24069M (SEQ ID NO.126), A24021M (SEQ ID NO.80), A24011M (SEQ ID NO.70), A24073M (SEQ ID NO.130), A24018M (SEQ ID NO.77), A24055M (SEQ ID NO.114), A24010M (SEQ ID NO.69), and A24065M (SEQ ID NO.79) show more than 80% knockdown of ANGPTL4 mRNA (equivalent to residual mRNA level of <0.2).

Example 15: Single-Dose Efficacy Screen of Intron-Targeting Mouse ANGPTL4-Specific Antisense Oligonucleotides in 4T1 Cells

2,500 4T1 cells/well were seeded in 96-well plates and treated with the ANGPTL4 antisense oligonucleotides (ASO) as shown in FIG. 15 at a final concentration of 5 μM. Three days after start of treatment, cell supernatant was replaced by fresh medium w/ASO and cells were incubated for additional 3 d. Then, cells were lysed and mouse Hprt1 and mouse ANGPTL4 mRNA expression was measured using the QuantiGene RNA Singleplex assay. ANGPTL4-mRNA expression values were normalized to expression of the housekeeping gene Hprt1. Residual ANGPTL4-mRNA expression relative to mock-treated cells (“no oligo” control set as 1) is shown. Solid line and dotted lines indicate 50% and 0% knockdown efficacy, respectively. Data are represented as mean of triplicate wells +/−SD.

As shown in FIG. 15 , six of the tested ANGPTL4 antisense oligonucleotides, which are A24047M (SEQ ID NO.106), A24095Mi (SEQ ID NO.151), A24093Mi (SEQ ID NO.149), A24020M (SEQ ID NO.79), A24090Mi (SEQ ID NO.146), and A24082Mi (SEQ ID NO.139) show more than 50% knockdown of ANGPTL4 mRNA (equivalent to residual mRNA level of <0.5).

Example 16: IC₅₀ Determination of Selected Mouse ANGPTL4-Specific Antisense Oligonucleotides

2,500 4T1 cells/well were seeded in 96-well plates and treated with the respective ANGPTL4 antisense oligonucleotides (ASO) A24018M (SEQ ID NO.77), A24019M (SEQ ID NO.78), A24020M (SEQ ID NO.79), A24021M (SEQ ID NO.80), A24047M (SEQ ID NO.106), A24054M (SEQ ID NO.113), A24065M (SEQ ID NO.79), A24070M (SEQ ID NO.127), A24072M (SEQ ID NO.129), A24082M (SEQ ID NO.139) and A24095Mi (SEQ ID NO.151) at different concentrations of 5000 nM, 1000 nM, 200 nM, 40 nM, 8 nM and 1.6 nM. Three days after start of treatment, cell supernatant was replaced by fresh medium w/ASO and cells were incubated for additional 3 d. Then, cells were lysed and mouse Hprt1 and mouse ANGPTL4 mRNA expression was measured using the QuantiGene RNA Singleplex assay. ANGPTL4-mRNA expression values were normalized to expression of the housekeeping gene Hprt1. Residual ANGPTL4-mRNA expression relative to mock-treated cells (“no oligo” control set as 1) is shown. Data are represented as mean of triplicate wells +/−SD.

FIG. 16 and Table 8 demonstrate that the selected ANGPTL4-specific antisense oligonucleotides inhibit ANGPTL4 mRNA expression dose-dependently with IC₅₀ values in the nanomolar range.

TABLE 8 IC+hd 50 +l values of selected mouse ANGPTL4- specific antisense oligonucleotides determined in 4T1 cells. ASO IC₅₀ [nM] R square A24018M 447 0.90 A24019M 122 0.87 A24020M 475 0.91 A24021M 283 0.68 A24047M 556 0.70 A24054M 350 0.85 A24065M 380 0.80 A24070M 329 0.59 A24072M  61 0.92 A24082Mi  56 0.65 A24095Mi 923 0.64

Example 17: 1^(st) Single-Dose Screen of Additional Mouse Angptl4-Specific ASOs in 4T1 Cells

Mouse Angptl4-specific antisense-oligonucleotides were designed. In the initial screen 53 ASOs targeting Angptl4 mRNA were tested. Three control oligonucleotides (R01002, R01014, Neg1) with different lengths (16, 17 and 18 nucleotides, respectively), that do not have sequence complementarity to any human or mouse mRNA were included as negative controls, whereas three ANGPTL4-specific oligonucleotides (A24047M (SEQ ID NO.106), A24072M (SEQ ID NO.129), A24095Mi (SEQ ID NO.151)) with verified knockdown efficiency were used as positive controls. Mouse breast cancer cells (4T1 cells) were treated with the respective oligonucleotides at a single concentration of 5 μM. After three days, cell supernatant was replaced by fresh medium containing 5 μM of the respective ASO and incubated for further three days. Afterwards, cells were lysed and mRNA levels were determined by QuantiGene RNA Singleplex assay. Hprt1 was used as a housekeeping gene for normalization of Angptl4 expression.

As shown in FIG. 17 , 15 ASOs (A24146Mi (SEQ ID NO.247), A24047M (SEQ ID NO.106), A24126Mi (SEQ ID NO.227), A24120Mi (SEQ ID NO.221), A24104M (SEQ ID NO.207), A24108M (SEQ ID NO.211), A24110M (SEQ ID NO.213), A24139Mi (SEQ ID NO.240), A24103M (SEQ ID NO.206), A24112M (SEQ ID NO.107), A24122HMe (SEQ ID NO.196), A24113M (SEQ ID NO.108), A24125Mi (SEQ ID NO.226), A24099M (SEQ ID NO.202), A24095Mi (SEQ ID NO.151)) reduced Angptl4 expression by more than 75% (equivalent to residual Angptl4-mRNA expression of <0.25), while control oligonucleotides (Neg1, R01002, R01014) reduced Angptl4 mRNA expression by less than 50% (equivalent to residual Angptl4-mRNA expression of >0.5) (FIG. 17 ).

Example 18: 2^(nd) Single-Dose Screen of Mouse Angptl4-Specific ASOs in Renal Cells

For further confirmation in a different cell line, the same treatment as in Example 17 was applied to mouse Renca cells. Thereby, treatment with 19 Angptl4-specific ASOs (A24143Mi (SEQ ID NO.244), A24047M (SEQ ID NO.106), A24095Mi (SEQ ID NO.151), A24125Mi (SEQ ID NO.226), A24110M (SEQ ID NO.213), A24148Mi (SEQ ID NO.151), A24120Mi (SEQ ID NO.221), A24104M (SEQ ID NO.207), A24109M (SEQ ID NO.212), A24139Mi (SEQ ID NO.240), A24103M (SEQ ID NO.206), A24122HMe (SEQ ID NO.196), A24131Mi (SEQ ID NO.232), A24138Mi (SEQ ID NO.239), A24123Mi (SEQ ID NO.224), A24146Mi (SEQ ID NO.247), A24117Mi (SEQ ID NO.218), A24130Mi (SEQ ID NO.231), A24144Mi (SEQ ID NO.245)) resulted in Angptl4 knockdown of more than 75% (equivalent to residual Angptl4-mRNA expression of <0.25) (FIG. 18 ).

Example 19: IC₅₀ Determination of Selected Mouse ANGPTL4-Specific Antisense Oligonucleotides

Based on results of knockdown efficiencies in 4T1 and Renca cells (FIG. 17, 18 ), nine ASOs (A24103M (SEQ ID NO.206), A24110M (SEQ ID NO.213), A24122HMe (SEQ ID NO.196), A24120Mi (SEQ ID NO.221), A24125Mi (SEQ ID NO.226), A24139Mi (SEQ ID NO.240), A24143Mi (SEQ ID NO.244), A24146Mi (SEQ ID NO.247), A24148Mi (SEQ ID NO.151)) with most potent knockdown efficacy Renca and 4T1 cells were selected for determination of half maximal inhibitory concentration (IC₅₀) values.

Primary mouse hepatocytes were treated with the respective ASO at different concentrations for three days. After three days, mRNA expression was analyzed using the QuantiGene Singleplex RNA assay. FIG. 19 and Table 9 demonstrate that the selected Angptl4-specific ASOs inhibit Angptl4 mRNA expression dose-dependently with IC₅₀ values in the nanomolar range.

Table 9 shows IC₅₀ values of selected Angptl4-specific ASOs determined in primary human hepatocytes. *, R square below 0.85; ASOs with increased potential to induce caspase3/7 leading to dose-dependent decrease of Hprt1 levels:

ASO IC₅₀ [nM] R square A24103M 159 0.90 A24110M 346 0.72* A24122HMe 117 0.98 A24120Mi 126 0.86 A24125Mi  70 0.91 A24139Mi 250 0.92 A24143Mi  60 0.84* A24146Mi  26 0.98 A24148Mi  88 0.97 A24047M  89 0.96 A24072M 118 0.96 A24095Mi  72 0.94

Conclusion Based on the Examples 7-11 and 17-19:

Testing a set of 47 further ASOs with specificity for human ANGPTL4, several ASOs were selected that potently reduce the expression of human ANGPTL4 in primary hepatocytes on the mRNA level. Treatment with 16 of the tested ASOs showed more than 70% knockdown of ANGPTL4 mRNA in primary human hepatocytes cells. Thereby, IC₅₀ values of most potent candidates were in low nanomolar range.

In order to conduct in vitro experiments in mouse models ASOs were designed with specificity for mouse Angptl4 and successful candidate ASOs were selected for in vivo studies that potently knock down mouse Angptl4 expression in vitro.

Taken together, a comprehensive set of in vitro experiments was conducted that led to the identification of highly potent human ANGPTL4 specific ASOs that are eligible for the development of an ASO-based ANGPTL4-targeting therapeutic. 

What is claimed is:
 1. A method of reducing ANGPTL4 expression in a subject, the method comprising administering a pharmaceutically active amount of an ANGPTL4 inhibitor consisting of an oligonucleotide of 12 to 22 nucleotides, wherein at least one nucleotide in the oligonucleotide is modified, and wherein the oligonucleotide is complementary to a corresponding portion of 12 to 22 consecutive nucleotides of positions 1264-1293 of SEQ ID NO:1 to the subject in need of the reducing.
 2. The method according to claim 1, wherein the subject has a cardiometabolic disease selected from the group consisting of obesity, diabetes, hypercholesterolemia, hypertriglyceridemia (HTG), dyslipidemia, pancreatitis, metabolic syndrome, and familial chylomicronemia syndrome (FCS).
 3. The method according to claim 2, wherein the disorder is a hypercholesterolemia selected from the group consisting of homozygous familial hypercholesterolemia (HoFH) and heterozygous familial hypercholesterolemia (HeFH).
 4. The method according to claim 2, wherein the disorder is type 2 diabetes.
 5. The method according to claim 1, wherein the oligonucleotide comprises the sequence (SEQ ID NO:186) (5′-3′) AGTACTGGCCGTTGAGG.
 6. The method according to claim 5, wherein the oligonucleotide comprises the sequence (5′-3′): +A*+G*+T*A*C*T*G*G*C*C*G*T*T*G*+A*+G*+G (SEQ ID NO:186), wherein + indicates a locked nucleic acid (LNA) nucleotide and * indicates a phosphorothioate (PTO) linkage between the nucleotides.
 7. The method according to claim 1, wherein the oligonucleotide of 12 to 22 nucleotides is a gapmer and comprises a central core of at least 5 consecutive deoxyribonucleotides and/or ribonucleotides.
 8. The method according to claim 1, wherein at least one of the modified nucleotides of the oligonucleotide is a 2′modified nucleotide or a bridged nucleotide.
 9. The method according to claim 5, wherein at least one of the modified nucleotides of the oligonucleotide is selected from a locked nucleic acid (LNA), cET, ENA, a 2′Fluoro modified nucleotide, a 2′O-Methyl modified nucleotide, 2′-Methoxyethyl modified nucleotide or a combination thereof.
 10. The method according to claim 1, wherein at least one of the modified nucleotides of the oligonucleotide is LNA.
 11. The method according to claim 1, wherein the oligonucleotide comprises a modified phosphate backbone.
 12. The method according to claim 1, wherein the oligonucleotide comprises a modified phosphate backbone, wherein the phosphate is a phosphorothioate.
 13. The method of claim 12, wherein the bridged nucleotide is a 2′-4′ bridged nucleotide. 